Renin inhibitors containing 5-amino-2,5-disubstituted-4-hydroxypentanoic acid residues

ABSTRACT

A series of novel polypeptide derivatives, containing 5-amino-2,5-disubstituted-4-hydroxypentanoic acid residues, which are useful for inhibiting the angiotensinogen-cleaving action of the enzyme renin. Particularly valuable precursors for many of these compounds are certain other 5-amino-2,5-disubstituted-4-hydroxypentanoic acid derivatives.

CROSS REFERENCE TO RELATED APPLICATION

This is a division, of application Ser. No. 07/336,697 U.S. Pat. No.4,948,913, filed on Nov. 2, 1987, which is a division of Ser. No.858,324, filed Apr. 30, 1986, now U.S. Pat. No. 4,729,985, which is acontinuation-in-part application of Ser. No. 764,168, filed Aug. 8,1985, now abandoned.

BACKGROUND OF THE INVENTION

The present invention is concerned with a series of novel polypeptidederivatives containing 5-amino-2,5-disubstituted-4-hydroxypentanoic acidresidues, which are useful for inhibiting the angiotensinogen-cleavingaction of the enzyme renin; and intermediates therefor, particularlyN-alpha-[N-(t-butoxycarbonyl)-phenylalanyl]-N(imidazole)-(t-butoxycarbonyl)histidine.

The proteolytic enzyme renin, which has a molecular weight of about40,000, is produced in and secreted into the blood by the kidney. It isknown to be active in vivo in cleaving the naturally-occurring plasmaglycoprotein angiotensinogen. In the case of human angiotensinogen,cleavage is at the bond between the leucine (10th) and valine (11th)amino acid residues at the N-terminal end of the angiotensinogen:Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val- ##STR1## The circulatingN-terminal decapeptide (angiotensin I) formed by the above cleavingaction of renin is subsequently broken down by the body to anoctapeptide known as angiotensin II. Angiotensin II is known to be apotent pressor substance, i.e. a substance that is capable of inducing asignificant increase in blood pressure, and is believed to act bycausing the constriction of blood vessels and the release of thesodium-retaining hormone aldosterone from the adrenal gland. Thus, therenin-angiotensinogen system has been implicated as a causative factorin certain forms of hypertension.

One means of alleviating the adverse effects of the functioning of therenin-angiotensinogen system is the administration of a substancecapable of inhibiting the angiotensinogen-cleaving action of renin. Anumber of such substances are known, including antirenin antibodies,pepstatin and naturally-occurring phospholipid compounds. EuropeanPatent Application No. 77,028 discloses a series of renin-inhibitingpolypeptide compounds having a non-terminal statine (Sta;4-amino-3-hydroxy-6-methylheptanoic acid) or statine derivative.Including Sta, the vast majority of compounds exemplified contain 6 ormore aminoacid residues. Exemplary of the few shortest chains theredisclosed are:

Acetyl-Phe-His-Sta-Leu-Phe-NH₂, and

t-Butyloxycarbonyl-Phe-His-Sta-Leu-Phe-NH₂.

There are invariably at least two amino acid residues each side ofstatine. The di- or polypeptidyl-statyl group is invariably attached toa lipophilic amino acid, most often leucine. (See also U.S. Pat. Nos.4,470,971 and 4,478,826.)

European Patent Application No. 45,665 and U.S. Pat. No. 4,424,207disclose a series of renin-inhibiting polypeptide derivatives of theformula ##STR2## where A is for example t-butoxycarbonyl, B is His orother basic aminoacyl group, D is Val, Ile or other liphophilicaminoacyl residue, E is Tyr, Phe, His or other aromatic aminoacylresidue, R^(a) and R^(b) are each isopropyl, isobutyl, benzyl or otherlipophilic aminoacid type sidechain, and Y^(a) is a terminal acid, esteror amide type group. Including the central 5-aminopentanoic acidresidues, these compounds are invariably heptapeptides, i.e.,N-tetrapeptidyl-5-aminopentanoyllipophilic aminoacyl-aromatic amino-acidderivatives.

SUMMARY OF THE INVENTION

We have now discovered that certain novel compounds possess exceptionalvalue as renin-inhibiting agents. These compounds are polypeptidederivatives of the formula ##STR3## or a pharmaceutically acceptablesalt thereof, wherein

n and m are each 1 or 0, the sum of n and m being at least 1;

W is ##STR4## where R⁵ is phenyl, 1-naphthyl, 4-hydroxyphenyl,4-methoxyphenyl, 4-imidazolyl, propyl or isopropyl;

W¹ is ##STR5##

W² is ##STR6## where R⁶ is --NH₂, ##STR7## or --CH₂ NH₂ ;

when n is 1, R is hydrogen, an amino-protecting acyl moiety having amolecular weight of less than 500, prolyl, pyroglutamyl, or prolyl orpyroglutamyl protected on nitrogen with said amino-protecting acylmoiety; and when n is O, R is phenoxyacetyl or2-benzyl-3-phenylpropionyl (dibenzylacetyl);

R¹ and R² are each independently hydrogen, (C₁ -C₆)alkyl, (C₁-C₆)alkenyl, phenyl, naphthyl, (C₄ -C₇)cycloalkyl, (C₄ -C₇)cycloalkenyl,(C₇ -C₉)phenylalkyl, (C₁₁ -C₁₃)naphthylalkyl, (C₅ -C₁₀)(cycloalkyl)alkyl, (C₅ -C₁₀) (cycloalkenyl)alkyl, or one of said groupsmono- or disubstituted on the aromatic ring with the same or differentgroups selected from (C₁ -C₃)-alkyl, (C₁ -C₃)alkoxy, fluoro or chloro;and

(a) R³ and R⁴ are taken separately, and are each independently hydrogen,(C₁ -C₆)alkyl, phenyl, naphthyl, (C₄ -C₇)cycloalkyl, adamantyl, (C₇-C₉)phenylalkyl, (C₁₁ -C₁₃)naphthylalkyl, (C₅ -C₁₀) (cycloalkyl)alkyl oradamantyl; or R³ is hydrogen and R⁴ is ##STR8##

p and q are each independently zero or an integer from 1 to 6;

r is 0 or 1;

Q is --CH₂ --, --CH═CH--, --O--, --NH--, --CHOH-- or --CO--;

Y is methyl, phenyl, --COOR⁹, --CONR⁹ R¹⁰, --CONHCOOCH₂ C₆ H₅, NH₂,--NHCOCH₂ C₆ H₅, ##STR9##

R⁷, R⁸, R⁹ and R¹⁰ are each independently hydrogen, (C₁ -C₆)alkyl,phenyl, (C₄ -C₇)cycloalkyl, (C₇ -C₉)-phenylalkyl, (C₅-C₁₀)(cycloalkyl)alkyl, or adamantyl; or

(b) R³ and R⁴ are taken together with the nitrogen to which they areattached to form a pyrrole, indoline, isoindoline, piperidine,1,2,3,4-tetrahydroquinoline, 1,2,3,4-tetrahydroisoquinoline,perhydroazepine, or morpholine ring system.

The expression "amino-protecting acyl moieties" refers to those acylgroups that are capable of substantially inhibiting reaction at thealpha-nitrogen of W (or W¹ when n=0) in vivo. R has a molecular weightof less than 500 in order to prevent an excessive detrimental effect onsolubility characteristics. Examples of suitable amino-protecting acylmoieties are well known to those skilled in the art, e.g. thet-butyloxycarbonyl, t-butylacetyl, benzyloxycarbonyl, t-butyluriedo,(tris-hydroxy)-(t-butyluriedo) and phenoxyacetyl moieties.

When n is 1 the preferred value of R is t-butoxycarbonyl, and thepreferred values of W and W¹ are phenylalanyl and histidyl,respectively. When m is 1, the preferred value of W² is lysyl. In allcases, the preferred value of R¹ is cyclohexylmethyl and the preferredvalues of R² are 2-methylpropyl, benzyl 2-methylpropenyl,4-chlorobenzyl, 4-methylbenzyl, 4-methoxybenzyl, 3,4-dichlorobenzyl,2-chlorobenzyl, 3-chlorobenzyl and cyclohexylmethyl, particularly2-methylpropyl. When m is 0 and n is 1, the preferred values of NR³ R⁴are NH₂, NHCH₃, N(CH₃)₂, NHCH₂ C₆ H₅, NH(CH₂)₃ COOH, NH(CH₂)₄ NH₂,NH(CH₂)₄ NHCOCH₂ H₆ H₅, and tetrahydroisoquinoline. When m and n areeach 1, the preferred values of NR³ R⁴ are phenylalanine and statineethyl ester.

The expression pharmaceutically acceptable salt refers to acid additionsalts of any of the compounds of the formula (I) which contain a basicfunctionality with an acid such as (but not limited to) HCl, HNO₃, H₂SO₄, H₃ PO₄, CH₃ SO₃ H, pCH₃ C₆ H₄ SO₃ H, CH₃ COOH or HOOCCH₂ CH₂ COOH.When the compound (I) contains more than one basic function, the saltoptionally contains more than one equivalent of acid. Alternatively,when the compound of the formula I contains an acidic function, theexpression also refers to cationic salts such as, but not limited to, analkali metal salt (e.g., Na, K), an alkaline earth salt (e.g., Ca, Mg)or an amine salt (e.g. diethanolamine, meglumine). Conventional methodsare used to prepare such salts.

The present invention also encompasses pharmaceutical compositionscontaining a renin inhibiting-effective amount of a compound of theformula (I) as the essential active ingredient in a pharmaceuticallyacceptable carrier; and a method for inhibiting the cleavage ofangiotensinogen by renin in the body of a mammal which comprisesadministering to the mammal an effective amount of a compound of theformula (I).

Additionally, the present invention includes intermediate compounds ofthe formula ##STR10## wherein

R¹ and R² are as defined above;

W³ is ##STR11## where R¹⁴ is --NHCO₂ CH₂ CH₂ C₆ H₅, ##STR12## or --CH₂NHCO₂ C₆ H₅ ;

R¹¹ is hydrogen or t-butoxycarbonyl; and

(a) R¹² and R¹³ are taken separately and are each independentlyhydrogen, (C₁ -C₆)alkyl, phenyl, naphthyl, (C₄ -C₇)cycloalkyl, adamantyl(C₇ -C₉)phenylalkyl, (C₁₁ -C₁₃)naphthylalkyl or (C₅ -C₁₀)(cycloalkyl)alkyl, or R¹² is hydrogen and R¹³ is ##STR13##

p, q, r and Q are as defined above;

Y¹ is methyl, phenyl, --COOR¹⁸, --CONR⁹ R¹⁰, --CONHCOOCH₂ C₆ H₅,--NHCOCH₂ C₆ H₅, ##STR14##

R⁷, R⁸, R⁹ and R¹⁰ are each independently as defined above; and

R¹⁸ is an independent value of R⁷ other than hydrogen; or

(b) R¹² and R¹³ are taken together with the nitrogen to which they areattached to form a pyrrole, indoline, isoindoline, piperidine,1,2,3,4-tetrahydroquinoline, 1,2,3,4-tetrahydroisoquinoline,perhydroazepine, or morpholine ring system; and

an intermediate compound of the formula ##STR15##

Finally, the present invention includes stereo-selective processes asfollows: ##STR16## where R¹ is as defined above, but is other thanhydrogen, and R¹⁵ is (C₁ -C₃)alkyl; and ##STR17## where X is chloro,bromo or another nucleophilically displaceable group, R¹ and R² are asdefined above, but R² is other than hydrogen and preferably anactivating allyl or benzyl type group; and the resultant, pure,heretofore unavailable chiral intermediates of the formulae type (D),(E), (F), (G) and (H) below. The preferred value of X is brom.

DETAILED DESCRIPTION OF THE INVENTION

The compounds of the invention are generally prepared by methodsfamiliar to those skilled in the art. The basic sub-unit of thepreferred chemical synthesis is the acylation of the unprotectedalpha-amino group of an amino acid residue with an amino acid having anactivated (for acylation purposes) carboxylic function and a suitableprotecting group bonded to its own alpha-nitrogen to form a peptide bondbetween the two amino acid residues, followed by the removal of saidprotecting group. This synthesis sub-unit of coupling-deblocking isperformed repeatedly to build up the polypeptide, starting from theC-terminal end of the molecular structure and working to the N-terminalend. The standard alpha-amino acids utilized to synthesize the compoundsof the present invention are commercially available (as free acids,salts or esters, etc.) in both alpha-amino protected and alpha-aminounprotected forms. Statine is commercially available asN-(t-butyloxycarbonyl)-statine; additionally, statine may be prepared(as a free acid or ester) in both the gamma-amino protected andgamma-amino unprotected forms by methods set forth in the literature(see e.g. U.S. Pat. No. 4,397,786 and Rich, D. H. et al., J. Org. Chem.vol. 43, pp. 3624 et seq. (1978). When desired, an appropriateN-unprotected amino acid analogue (free acid, salt or ester, etc.) suchas 4-aminobutyric acid, 4-aminovaleric acid,5-(benzyloxyoxycarbonylamino)pentylamine or ethyl3-(2-aminoethoxy)-propionate is used as a reactant in the first couplingstep. If not commercially available or previously known, such compoundsare readily prepared by methods well known in the art of organicchemistry. When the desired product contains a basic amino acid residueW² (i.e. m=1), the required intermediate H-(W³)--NR¹² R¹³ (wherein W³,R¹² and R¹³ are defined above) is prepared from lysine, ornithine orarginine, protected on the alpha-NH₂ group with a hydrolyzable groupsuch as t-butoxycarbonyl (t-Boc) which can be cleaved under anhydrousacid conditions (e.g. ca. 3-5N HCl in dioxane, or anhydroustrifluoroacetic acid) and on omega-nitrogen with a hydrogenolyzablegroup such as benzyloxycarbonyl (cbz), by coupling with an amine HNR¹²R¹³. Preferred coupling conditions employ more or less equimolarquantities of the reagents to be coupled, dicyclohexylcarbodiimide (or asimilar dehydrative coupling agent) in an equimolar quantity and1-hydroxybenzotriazole in 1-2 molar quantity, in a reaction inertsolvent such as CH₂ Cl₂ at 0°-50° C. (conveniently at ambienttemperature). When a reactant is in the form of an acid salt, a tertiaryamine (e.g., triethylamine, N-methylmorpholine) is employed in an amountsufficient to neutralize said acid. Prior to the next coupling step thealpha-amino group of the basic amino acid residue W² is deprotected,e.g., an alpha-t-Boc group is readily removed by the action of 3.5 to4.5N HCl in dioxane at -10° to 40° C., conveniently at ambienttemperature; or by the action of anhydrous trifluoroacetic acid at -20°to 20° C., conveniently at about 0°-5° C.

The required 2,5-disubstituted-4-hydroxypentanoic acid residues mustgenerally be synthesized. A present stereoselective route is from aprecursor of appropriate chirality, conveniently an L-amino acid, e.g.,in the initial stages: ##STR18## The starting protected L-amino acid isobtained commercially or by standard methods well known in the art,including, when desired, reduction of an aromatic ring, e.g., that ofN-(t-butoxycarbonyl)phenylalanine methyl ester as exemplified below. Thelower alkyl ester, preferably the methyl ester as shown is readilyreduced to the aldehyde, for example, with diisobutylaluminum hydride intoluene at -50° to -80° C. The aldehyde in turn is reacted withLiC.tbd.CCOOR¹⁵ (usually R¹⁵ is ethyl formed in situ from ethylpropiolate), again at -50° to -80° C., in a reaction inert solvent suchas tetrahydrofuran. A pair of diastereoisomers are generally formed atthis stage, with the desired diastereoisomer (C) greatly predominating.The lesser, undesired isomer is preferably removed followinghydrogenation of the triple bond (carried out under standardhydrogenation conditions, e.g., over a palladium catalyst, preferably Pdin BaSO₄ under relatively mild conditions; and formation of the lactone(e.g., by reacting in toluene in the presence of acetic acid).

The desired lactone epimer, having the 4S stereochemistry shown informula (E) and (F), is then condensed with a halide, R² X (X=Cl, Br orI; preferably X=Br) in the presence of a substantial excess, e.g., 2 to2.5 molar equivalents of a strong base of low nucleophilicity, such asLiN[CH(CH₃)]₂ or preferably, lithium hexamethyldisilazide. Preferablythe halide is an allylic or benzylic type halide (e.g.,2-methyl-2-propenyl bromide, benzyl bromide) with the double bond oraromatic ring subsequently hydrogenated if the saturated group R² isdesired, e.g., ##STR19## Once again, the desired diastereoisomer, havingtrans (2R) stereochemistry as shown, predominates. It is separated bychromatography (before or after any required hydrogenation to producethe desired group R²). Alternatively, hydrogenation, particularly whenR² at the stage of condensation is a benzyl group, can be deferred untillatter in the overall synthesis of the desired compound. The preferredcatalysts for hydrogenation of a simple olefin comprise Pd or Rh, whilefor reduction of phenyl to cyclohexyl, Rh is preferred.

In certain preferred embodiments of the present invention, particularlywhen NR¹² R¹³ is simple amine, such as NH₂, NHCH₃, N(CH₃)₂ or NHCH₂ C₆H₅, the lactone is reacted directly with the appropriate amine, e.g.,##STR20##

The lactones react smoothly with excess of lower molecular weight, morebasic amines such as NH₃, NH₂ CH₃ and NH(CH₃)₂ at lower temperatures(e.g., 0° to 40° C.) in a reaction inert solvent. With more hindered orless basic amines, higher temperatures e.g., 80°-100° C., are employed,optionally in the presence of an acetic acid catalyst.

With more complex NR¹² R¹³ groups, it is preferred to convert lactone,via saponification of the blocked lactone (J) to acid with dilute NaOHin an aqueous solvent, conversion of the acid to benzyl ester usingbenzyl bromide in the presence of K₂ CO₃ in a reaction inert solventsuch as dimethylformamide, formation of tetrahydropyranyl ether by theaction of 3,4-dihydro-2H-pyran in a reaction-inert solvent in thepresence of a catalyst such as pyridinium p-tosylate and finallyhydrogenolysis to the acid: ##STR21## The latter is then coupled with anamino compound

    H--(W.sup.3).sub.m --NR.sup.12 R.sup.13

using the dehydrative coupling method (e.g., withdicyclohexylcarbodiimide) which is described above, or the so-calledmixed anhydride procedure, also well known in the art and exemplifiedbelow; followed by selective hydrolysis of the tetrahydropyranyl etherby warming in aqueous acetic acid to produce the intermediate compoundof the formula (II) wherein R¹¹ is t-butoxycarbonyl.

The compound of the formula (II) wherein R¹¹ is t-Boc, regardless of itssource, is then selectively cleaved (by the methods described above) toyield the free amino compound (II) wherein R¹¹ is hydrogen.

In the final stages of the preferred process, the intermediate iscoupled with

    R--W.sup.4 --(W.sup.5).sub.n --OH

wherein R and n are as defined above; and W⁴ and W⁵ correspond to aboveW and W¹ respectively, but with imidazole nitrogen of any histidineresidue blocked with t-butoxycarbonyl. The preferred coupling methodemploys dicyclohexylcarbodiimide using conditions as described above.This preferred method shows particular advantage, when n is 1, over theusual method of introducing each amino acid in single, sequential steps.Finally, any imidazole t-boc protecting group is selectively removed byhydrolysis in aqueous acetic acid (e.g., 80% acetic acid) at 10°-40° C.,and any N-benzyloxycarbonyl or benzyl ester blocking groups areselectively removed by standard hydrogenation methods as describedabove, leaving the generally desired N-terminal protecting group intact.

The activity of the compounds of the present invention as inhibitors ofthe angiotensinogen-cleaving activity of renin is determined by studying(1) their ability to inhibit the angiotensinogen-cleaving activity ofrenin in vitro. In this test, blood plasma is obtained from healthylaboratory personnel, pooled and stored frozen until required. Beforeuse, a quantity of this plasma is defrosted and centrifuged, and thesupernatant mixed with protease inhibitors and buffered to pH 7.4. Renininhibitors are added at different levels to different aliquots of theplasma supernatant, and the resulting mixtures (310 lambda) incubatedfor three hours at 37° C. along with renin inhibitor-free controlmixtures. After incubation, the mixtures are quenched in ice water andeach assayed for angiotensin I using angiotensin I antibody. Theproduction of angiotensin I in the presence of a renin inhibitor iscompared to that produced in the absence of the inhibitor, and apercentage inhibition is calculated. Using data obtained from duplicateincubations at each of several difference inhibitor concentrations, theinhibitor concentration in the incubation mixture required to produce afifty percent inhibition of the angiotensinogen-cleaving activity ofrenin, i.e. the IC₅₀ of the inhibitor, is calculated for variousdifferent inhibitors. The angiotensin I in the quenched incubationmixtures is assayed by means of a radioimmunoassay, using components ofa renin radioimmunoassay kit supplied by Becton Dickinson and Co.(Orangeburg, N.Y.). This radioimmunoassay was based upon the onedeveloped by Haber et al., J. Clin. Endocrinol., 29, pp. 1349-1355(1969).

The activity of the present compounds may also be determined by theirability to antagonize the exogenous renin-induced pressor response invivo. Male Sprague-Dawley rats (230 to 300 g. body weight) areanesthetized with sodium pentobarbital (65 mg./kg. body weight,intraperitoneal), after which femoral vein and carotid artery cathetersare implanted in each animal. Following completion of surgery, theanimals are placed in the prone position and rectal temperaturemonitored continuously. Mean arterial blood pressure (MAP) is recordedvia the carotid artery catheter using a Statham P23 ID pressuretransducer and a physiograph. Subsequent to a stabilization period,control renin pressor responses (dP) of 20 to 30 mm Hg are obtained byadministration of hog renin (30 to 80 mU/kg. body weight, intravenous),the animals are rechallenged with hog renin (same dosage as for controlresponse) at 5, 15 and 30 minutes after renin inhibitor administrationand the corresponding renin pressor responses (dP) measured. Percentantagonization is calculated as ##EQU1## where control dP andexperimental dP are the pressor changes in MAP before and after renininhibitor administration, respectively. Preferably, at least threeanimals are used in each test, with results averaged

The compounds of the present invention can be administered asantihypertensive agents by either the oral or parental routes ofadministration, and are routinely effective by the latter route,particularly when dosed as an intravenous solution Wheregastrointestinal adsorption permits, oral administration is preferredfor reasons of patient convenience and comfort. In general, theseantihypertensive compounds are normally administered in dosages rangingfrom about 0.1 mg. to about 10 mg. per kg. of body weight per day;variations will necessarily occur depending upon the condition of thesubject being treated and the particular compound being administered.Typically, treatment is commenced at a low daily dosage and increased bythe physician only if necessary. It is to be noted that these compoundsmay be administered in combination with pharmaceutically acceptablecarriers by either of the routes previously indicated, and that suchadministration can be carried out in both single and multiple dosages.

For parenteral use, the present compounds are formulated according tothe known art using suitable dispersing or wetting agents and suspendingagents. The sterile injectable formulation can also be a solution ofsuspension in a non-toxic parenterally acceptable diluent or solvent,for example, as a solution in 1,3-butandiol. Among the acceptablevehicles and solvents are water, Ringer's solution and isotonic NaClsolution, fixed oils including synthetic mono- or diglycerides, fattyacids such as oleic acids, and mixtures thereof.

For oral administration, a wide variety of dosage forms are used, e.g.,tablets, capsules, lozenges, troches, hard candies, powders, sprays,aqueous suspension, elixirs, syrups, and the like formulated withvarious pharmaceutically-acceptable inert carriers. Such carriersinclude solid diluents or fillers, sterile aqueous media and variousnon-toxic organic solvents, etc. In general, the compounds of thisinvention are present in such oral dosage forms at concentration levelsranging from about 0.5% to about 90% by weight of the total composition,in amounts which are sufficient to provide the desired unit dosage.Tablets may contain various excipients such as sodium citrate, calciumcarbonate and calcium phosphate, along with various disintegrants suchas starch (preferably potato or tapioca starch), alginic acid andcertain complex silicates, together with binding agents such aspolyvinylpyrrolidione, sucrose, gelatin and acacia. Additionally,lubricating agents such as magnesium stearate, sodium lauryl sulfate andtalc are often very useful for tabletting purposes. Solid compositionsof a similar type may also be employed as fillers in soft andhard-filled gelatin capsules; preferred materials in this connectionwould also include lactose or milk sugar as well as high molecularweight polyethylene glycols. When aqueous suspensions and/or elixirs aredesired for oral administration, the essential active ingredient thereinmay be combined with various sweetening or flavoring agents, coloringmatter or dyes and, if so desired, emulsifying and/or suspending agentsas well, together with such diluents as water, ethanol, propyleneglycol, glycerin and various like combinations thereof.

All structural designations of stereochemistry shown herein representabsolute stereochemistry. The present invention is illustrated by thefollowing examples. However, it should be understood that the inventionis not limited to the details of these examples. All temperatures are in°C. and are ambient unless otherwise specified. All stripping ofsolvents was in vacuo. All standard solutions are in water unlessotherwise specified. THF stands for tetrahydrofuran; DMF stands fordimethylformamide; DME stands for 1,2-dimethoxyethane; and DMSO standsfor dimethylsulfoxide. All standard alpha-amino acids or derivatives arein the L-form. Statine is 3S,4S-4-amino-3-hydroxy-6-methylheptanoicacid.

EXAMPLE 1 S-3-Cyclohexyl-2-(t-butoxycarbonylamino)-propionaldehyde

Methyl S-3-cyclohexyl-2-(t-butoxycarbonylamino)-propionate (51.2 g.,0.179 mol) was dissolved in 728 ml. of dry toluene and cooled to -78°.Diisobutylaluminum hydride (449 ml. of 1M in toluene, 0.449 mol) wasadded dropwise over 1 hour, maintaining -70° to -78°. Methanol (13 ml.)was added at -70°, followed by 608 ml. of half-saturated sodiumpotassium tartrate, and the mixture warmed to ambient temperature. Ether(300 ml.) was added and the organic layer was separated and washed with1 1. saturated sodium potassium tartrate. The original aqueous layer wasextracted with 600 ml. fresh ether and backwashed with 600 ml. freshsaturated sodium potassium tartrate. The organic layers were combined,dried over MgSO₄ and stripped to yield title product as a gum,contaminated with toluene on the basis of ¹ H-nmr, 45.6 g; tlc Rf 0.45(1:3 ethyl acetate:hexane); ¹ H-nmr (CDCl₃) delta: 0.9 to 2.3 (m), whichincludes t-butyl singlet at 1.4, 3.0-4.8 (m), 4.9-5.2 (d), 9.6 (s).

EXAMPLE 2 Ethyl4RS,5S-6-Cyclohexyl-5-(t-butoxy-carbonylamino)-4-hydroxy-2-hexynoate

Dry freshly distilled THF (117 mol) and diisopropylamine (22.0 ml., 15.8g., 0.156 ml.) were charged to a flame dried reaction flask under N₂ andthe resulting solution cooled to -30° and butyllithium (76.9 ml. of 1.6Min hexane, 0.123 mol) added over 5 minutes. The solution was then cooledto -78° and ethyl propiolate (12.5 ml., 12.1 g., 0.123 mol) addeddropwise over 20 minutes, maintaining the temperature -65° to -78°.After 30 minutes at -78° , title product of the preceding Example (19.52g., 0.0866 mol) in 35 ml. THF was added over 20 minutes, againmaintaining -65° to -78°. After 2 hours, 200 ml. of 5:1 THF:acetic acidwas added to the reaction mixture, and it was allowed to warm to ambienttemperature and diluted with a half volume of ether and an equal volumeof 10% citric acid.

The organic layer was separated, washed sequentially with 2×200 ml.fresh 10% citric acid, 200 ml. of brine and 2×200 ml. saturated NaHCO₃,dried over MgSO₄ and stripped to a dark red oil, 38.2 g. The latter waschromatographed on a 10 cm×42 cm column of silica gel with tlcmonitoring, eluting with 5 l. of 1:9 ethyl acetate:hexane. After 1500ml. to develop the column, 500 ml. fractions were collected. Fractions29-37 were combined and stripped to yield title product as an oil, 15.3g.; tlc Rf 0.44 (3:7 ethyl acetate:hexane); ¹ H-nmr CDCl₃) delta:1.0-2.0 (m, 25H) including singlet for the t-butyl group at 1.5, 3.8-5(m, 6H).

EXAMPLE 34S,5S-6-Cyclohexyl-5-(t-butyloxycarbonylamino)-gamma-hexanolactone

Title product of the preceding Example (18.28 g.) and 5% Pd/BaSO₄ (10.97g.) were combined with 150 ml. ethyl acetate and hydrogenated for 2hours under 4 atmospheres pressure of hydrogen. The catalyst wasrecovered by filtration and the filtrate stripped to yield intermediateethyl 4RS,5S-6-cyclohexyl-4-hydroxy-5-(t-butyloxyamino)hexanoate, 19 g.The latter was taken up in 250 ml. of 2.5% acetic acid in toluene,refluxed 2.5 to 3 hours, stripped and the residue chromatographed on a10 cm.×30 cm. column of silica gel, monitoring by tlc, eluting with 4 l.of 9:11 ether:hexane, 8 l. of 1:1 ether:hexane, 2 l. of 11:9ether:hexane and finally 3 l. of 3:2 ether:hexane, collecting 28×400 ml.fractions, 6×150 ml. fractions and finally 11×400 ml. fractions.Fractions 17-24 were combined and co-stripped with ether to yield thepredominant and desired, less polar, 4S,5S-title product as an oil, 9.13g.; tlc Rf 0.5 (7:3 ether hexane). The more polar, 4R,5S-epimer of titleproduct was isolated by stripping combined fractions 28-45 andcrystallized by trituration with hexane, 1.77 g.; mp 101.5°-103.5°.

EXAMPLE 4 2R,4S,5S- and2S,4S,5S-6-Cyclohexyl-5-(t-butoxycarbonylamino)-2-(2-methyl-2-propenyl)-gamma-hexanolactone

Dry freshly distilled THF (30 ml.) and diisopropylamine (3.51 ml., 2.52g., 0.0249 mol) were charged to a flame dried reaction flask under N₂,the resulting solution was cooled to -50°, butyllithium (13.9 ml. of1.6M in hexane, 0.0222 mol) was added and the mixture further cooled to-78°. Title product of the preceding Example (2.77 g., 0.0089 mol) in 15ml. THF was added dropwise over 10 minutes and the enolate allowed toform over a further 20 minutes at -78°, at which time3-bromo-2-methyl-1-propene in 5 ml. THF was added over 10 minutes, andthe mixture stirred an additional 1 hour at -78°, quenched with 5 ml.saturated NH₄ Cl, warmed to room temperature, diluted with a half volumeof ether, washed 2×50 ml. 10% citric acid, 2×50 ml. saturated NaHCO₃ and1×25 ml. brine, dried over MgSO₄ and stripped to an oil, 3.06 g., amixture of the title epimers. The latter were separated bychromatography on 7 cm×20 cm silica gel; monitoring by tlc; elutingsequentially with 2 l. of 1:9 ether:hexane, 4 l. of 3:17 ether:hexane, 2l. of 1:4 ether:hexane, 2 l. of 1:3 ether:hexane, 2 l. of 7:13ether:hexane and 2 l. of 1:1 ether hexane; and collecting 125 ml.fractions. The less polar title product, having trans (2R)stereochemistry, was collected in fractions 30-48, combined and strippedto yield same as an oil, 1.17 g.; tlc Rf 0.45, (2:3 ether:hexane); ^(H)-nmr (CDCl₃) delta 1.4 (s, 9H), 1.8 (s, 3H), 0.3-3.0(m, 18H), 3.6-4.0(m, 1H), 4.69 (s, 1H), 4.75 (s, 1H), 4.1-4.8 (m, 2H). Fractions 55-76gave the more polar title product, also as an oil, 0.358 g., having cis(2S) stereochemistry; tlc Rf 0.36 (2:3 ether:hexane); ¹ H-nmr identicalto that of the less polar epimer.

EXAMPLE 5 2R,4S,5S- and2S,4S,5S-6-Cyclohexyl-5-5-(t-butoxycarbonylamino)-2-(2-methylpropyl)-gamma-hexanolactone

The less polar title product of the preceding Example (1.17 g.) and 10%Pd/C (0.351 g.) were combined in 20 ml. ethyl acetate and hydrogenatedat 4 atmospheres pressure for 2.5 hours, the catalyst recovered byfiltration and the filtrate stripped to yield less polar title product(likewise having trans, i.e., 2R stereochemistry) as an oil whichcrystallized on standing, 1.20 g.; mp 88°-93°; tlc Rf 0.65 (1:1 ether:hexane), Rf 0.73 (2:1 ethyl acetate:hexane). The other isomer, havingcis (2S) stereochemistry, was obtained in like manner; tlc Rf 0.59 (1:1ether:hexane). In subsequent Examples, which employ the present lesspolar epimer of Rf 0.65 (1:1 ether:hexane), 2R stereochemistry isspecified.

EXAMPLE 6 Benzyl2R,4S,5S-6-Cyclohexyl-5-{t-butoxycarbonylamino)-4-hydroxy-2-(2-methylpropyl)-hexanoate

The less polar 2R title product of the preceding Example (1.0 g., 2.72mmols), DME (14 ml.) and NaOH (4.4 ml. of 5N, 0.022 mol) were combined,stirred for 12 hours and then stripped of organic solvent. The aqueousresidue was diluted with water (5 ml.), layered with an equal volume ofether, cooled to 0° and acidified with 22 ml. of 1N HCl, by which timethe aqueous layer was turbid. The ether layer was separated and theaqueous layer (pH 2.0) washed with an equal volume of fresh ether. Theether layers were combined, dried over MgSO₄ and stripped to yieldintermediate2R,4S,5S-6-cyclohexyl-5-(t-butoxycarbonylamino)-4-hydroxy-2-(2-methylpropyl)hexanoicacid as a white foam, 0.909 g. (2.36 mmols). The latter was charged into20 ml. DMF together with K₂ CO₃ (0.330 g., 2.36 mmols) and cooled to 0°.Benzyl bromide (0.308 ml., 0.443 g., 2.59 mmols) was added and thestirred mixture allowed to warm to room temperature. After 4 hours, thereaction mixture was diluted with an equal volume of ether, washed 1×75ml. 1% citric acid, 1×75 ml. saturated NaHCO₃, dried over MgSO₄, andstripped to yield an oil, 1.35 g. The latter was chromatographed on 4cm×20 cm silica gel; monitored by tlc; eluting with 1 l. 1:9 etherhexane, 1 l. 3:17 ether: hexane, 1 l. 1:4 ether hexane and finally 1 l.3:7 ether:hexane; and collecting 23 ml. fractions. Fractions 46-75 werecombined and stripped to yield crystalline starting material, 0.583 g.,suitable for recycling. Fractions 85-119 were combined and stripped toyield present title product as an oil, 0.275 g.; tlc Rf 0.47 (1:1ether:hexane); ¹ H-nmr CDCl₃): 0.3-2.0 (m, 24H), 1.4 (s, 9H), 2-2.4 (m,1H), 2.6-3.0 (m, 1H), 3.2-3.7 (m, 2H), 4.65 (d, 1H), 5.1 (s, 2H), 7.3(s, 5H).

EXAMPLE 7 Benzyl2R,4S,5S-6-Cyclohexyl-5-(t-butoxycarbonylamino)-2-(2-methylpropyl)-4-(2-tetrahydropyranyloxy)hexanoate

The product of the preceding Example (0.442 g., 0.929 mmol),3,4-dihydro-2H-pyran (97%, 0.255 ml., 0.235 g., 2.79 mmols) andpyridinium p-tosylate (23 mg., 0.093 mmol) were combined in 6.5 ml. CH₂Cl₂ and stirred in a stoppered flask for 18 hours, then stripped ofsolvent and the residue taken up in 100 ml. ether, washed 2×100 ml. halfsaturated brine, dried over MgSO₄, co-stripped with CH₂ Cl₂, and driedunder high vacuum to yield present title product as an oil, 0.565 g.;tlc Rf 0.59 (2:3 ether:hexane).

EXAMPLE 82R,4S,5S-6-Cyclohexyl-5-(t-butoxycarbonylamino)-2-(2-methylpropyl)-4-(2-tetrahydropyranyloxy)-hexanoicAcid

The product of the preceding Example (0.565 g.) and 10% Pd/C (0.170 g.)were combined in 10 ml. ethyl acetate and hydrogenated at 4 atmospherespressure for 2.5 hours. The catalyst was recovered by filtration and thefiltrate co-stripped with CH₂ Cl₂ to yield title product as an oil, 0.55g., which contains the theoretical yield (0.474 g.) of product; tlc Rf0.0 (2:3 ether:hexane), 0.35 (1:1 ethyl acetate:hexane); ¹ H-nmr (CDCl₃)includes 9.5-10.1 (bs, 1H, COOH) and no peaks due to a benzyl group.

This example was repeated on a 300 mg. scale with drying of the productunder high vacuum to produce 250.5 mg. (a quantitative yield of titleproduct as a white foam).

EXAMPLE 9N-[N-alpha-(t-Butoxycarbonyl)-N-epsilon-(benzyloxycarbonyl)lysyl]phenylalanineBenzyl Ester

N-hydroxybenzotriazole (162 mg., 1.2 mmoles), N-methylmorpholine (101.2mg., 1 mmole), L-phenylalanine benzyl ester p-toluenesulfonate (428 mg.,1 mmole),N-alpha-(t-butyloxycarbonyl)-N-epsilon-benzyloxycarbonyl-L-lysine (456mg., 1.2 mmoles) and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimidemetho-p-toluene sulfonate (635 mg, 80% pure, 1.2 mmoles) weresequentially dissolved in methylene chloride (50 ml.) at 0° C., and theresulting solution stirred for 19 hours at 20° C. The reaction mixturewas then washed consecutively with 75 ml of 5.5% aqueous HCl, 75 ml. ofsaturated aqueous NaHCO₃ and 75 ml. of brine, dried over MgSO₄, andstripped to yield title product as a dry foam, 0.699 g.; ¹ H-nmr (CDCl₃)includes delta 1.5 (s, 9H, t-butyl). The product was used directly inthe next Example, without further purification.

EXAMPLE 10 N-[N-epsilon-(Benzyloxycarbonyl)lysyl]phenylalanine BenzylEster Hydrochloride

Product of the preceding step (0.650 g.) was dissolved in 7 ml. of 3.7NHCl in dioxane, allowed to stand 1 hour, and stripped to yield titleproduct, 0.583 g.; ¹ H-nmr (CD₃ OD) includes delta 5.2 (s, 2H, --CH₂ C₆H₅).

EXAMPLE 11N-(N-alpha-[2R,4S,5S-6-Cyclohexyl-5-(t-butoxycarbonylamino)-4-hydroxy-2-(2-methylpropyl)hexanoyl]-N-epsilon-(benzyloxycarbonyl)lysyl))-phenylalanineBenzyl Ester Hydrochloride

The product of the preceding Example (0.560 g., 1.01 mmols) was stirredwith 5 ml. CH₂ Cl₂ and cooled to 0° under nitrogen. Triethylamine (0.211ml., 1.51 mmols) and then the product of Example 8 (0.474 g., 1.01mmols) in 5 ml. CH₂ Cl₂ were added. Finally, 1-hydroxybenzotriazole(0.204 g., 1.51 mmoles) and then dicyclohexylcarbodiimide (0.208 g.,1.01 mmols) in 5 ml. CH₂ Cl₂ were added. The reaction mixture wasallowed to warm to ambient temperature and stirred 18 hours. Theresulting suspension was filtered and the filtrate stripped of CH₂ Cl₂,diluted with 15 ml. ethyl acetate, and refiltered. The second filtratewas washed 2×10 ml. 10% citric acid, 2×10 ml. saturated NaHCO₃ and 1×10ml. brine, dried over MgSO₄ and co-stripped with CH₂ Cl₂ to yieldintermediate tetrahydro-2-pyranyl ether of title product, 0.97 g. Thelatter was stirred for 20 hours with 35 ml. 70% acetic acid. The mixturewas stripped to low volume and the residue co-stripped with toluene toproduce a solid, white residue. The latter was taken into 20 ml. CH₂Cl₂, washed 1×10 ml. brine and 2×10 ml. saturated NaHCO₃, dried overMgSO₄, and stripped to yield white solids, 1.02 g., which werechromatographed on 7 cm×15 cm silica gel eluting with 2 l. 0.5% methanolin CHCl₃, monitoring by tlc and collecting 125 ml fractions. Titleproduct was recovered as a white solid by stripping combined fractions29-40 and triturating the residue with ether, 0.497 g.; mp 157°-159°;tlc Rf 0.39 (1:1 ethyl acetate:hexane); ¹ H-nmr (CDCl₃) includes 1.4 (s,9H, t-butyl) and 6.5-7.4 (m, 15H, aromatic).

EXAMPLE 12N-alpha-((2R,4S,5S-6-Cyclohexyl-5-[(N-t-butoxycarbonylphenylalanyl)histidyl]amino-4-hydroxy-2-(2-methylpropyl)hexanoyl))-N-epsilon-((benzyloxycarbonyl))lysyl-phenylalanineBenzyl Ester

Product of the preceding Example (250 mg., 0.282 mmol) was stirred with3 ml. trifluoroacetic acid, protected from moisture by a CaCl₂ dryingtube, and cooled to 0°. After 35 minutes the reaction was co-strippedwith ether to yield intermediateN-((N-alpha-[2R,4S,5S-6-cyclohexyl-5-amino-4-hydroxy-2-(2-methylpropyl)hexanoyl]-N-epsilon-(benzyloxycarbonyl)lysyl))phenylalaninebenzyl ester trifluoroacetate salt as a white foam, 0.291 g. The latter,under N₂ was taken into 2 ml. CH₂ Cl₂, cooled to 0° and triethylamine(0.0471 ml., 0.0342 g., 0.338 mmol), 1-hydroxybenzotriazole (0.0571 g.,0.423 mmol),N-alpha-[N-(t-butoxycarbonyl)phenylalanyl]-N(imidazole)-(t-butoxycarbonyl)histidine(149.7 mg., 0.296 mmol) and dicyclohexylcarbodiimide (61.1 mg., 0.296mmol) in 5 ml. CH₂ Cl₂ were added sequentially. The reaction mixture wasallowed to warm to room temperature, stirred for 18 hours, the resultingsuspension stripped of solvent, the residue suspended in 5 ml. ethylacetate, filtered to removed dicyclohexyl urea, yielding 373 mg. ofsolids, determined by tlc to also contain considerable amounts of thedesired intermediate product retaining the histidineimidazole-(tbutoxycarbonyl) protecting group on gamma histidine,nitrogen; tlc Rf 0.51 (1:19 methanol:CHCl₃). The filtrate was strippedto 140 mg. residue containing additional intermediate by tlc and ¹ H-nmr(CDCl₃) showing two singlets at 1.3 and 1.6 for the two t-butyl groupsand a singlet at 5.1 for the methylene of the benzyl group. Bothintermediate containing materials were combined and treated, withstirring under nitrogen, with 70 ml. of saturated dimethylamine in CHCl₃for 3 hours. The reaction mixture was then stripped to solids free ofamine odor, and chromatographed on 7 cm×15 cm of silica gel, gradientlyeluting with 2 to 5% methanol in CHCl₃ and monitoring by tlc. Productfractions were combined, stripped and triturated with hexane/ether toyield title product as a white solid, 0.182 g.; tlc Rf 0.85 (1:1:1:1ethyl acetate:butanol: acetic acid:H₂ O), Rf 0.24 (1:19 methanol:CHCl₃);¹ H-nmr (CDCl₃ with minor portion of CH₃ OD) included 1.3 (s, 9H,t-butyl), 6.8 (s, 1H) and 7.5 (s, 1H)-imidazole protons, and 5.0 (s, 2H,CH₂ C₆ H₅).

EXAMPLE 13 N-alpha-((2R,4S,5S-6-Cyclohexyl-5-[N-(t-butoxycarbonylphenylalanyl)histidyl]amino-4-hydroxy-2-(2-methylpropyl)hexanoyl))lysyl-phenylalanineDiacetate

Product of the preceding Example (70 mg.) and 10% Pd/C (70 mg.) werecombined with 10 ml. 4:1 methanol: acetic acid and hydrogenated at 4atmospheres for 2 hours. The catalyst was recovered by filtration andthe filtrate co-stripped with toluene to yield title product as a whitepowder, 73 mg.; tlc Rf 0.63 (1:1:1:1 ethyl acetate:butanol:aceticacid:H₂ O), Rf 0.0 (1:19 methanol:CHCl₃); ¹ H-nmr (DMSO-d ) includes 1.3(s, 9H, t-butyl), no benzyl CH₂ Cl₂ protons and aromatic protons at6.8-7.8 ppm; mp, decompose above 200°.

EXAMPLE 142R,4S,5S-6-Cyclohexyl-5-(t-butoxycarbonylamino)-4-hydroxy-2-(2-methylpropyl)-N-methylhexanamide(Method A)

2R,4S,5S-6-Cyclohexyl-5-t-butoxycarbonylamino)-2-(2-methylpropyl)-gamma-hexanolactone(197 mg., 0.536 mmol, Example 5) was dissolved in 1 ml. of water andcooled in an ice-water bath. The cold solution was perfused with CH₃ Cl₂for 3 minutes, stoppered and allowed to stand at room temperature for 2hours, by which time tlc indicated reaction was complete. The mixturewas stripped of solvent and dried under high vacuum to yield titleproduct as a white, solid foam, 204 mg.; tlc Rf 0.31 (3:1 ethylacetate:hexane).

EXAMPLE 152R,4S,5S-6-Cyclohexyl-5-amino-4-hydroxy-2-(2-methylpropyl)-N-methylhexanamideHydrochloride

Title product of the preceding Example (199 mg., 0.499 mmol) wasdissolved in 4N HCl in dioxane (1 ml.) and stirred for 25 minutes underNhd 2, by which time tlc indicated complete consumption of startingmaterial. The reaction mixture was then stripped of solvent and dried inhigh vacuum to produce title product as an incompletely dry, pale yellowpowder, 185 mg.; tlc Rf 0.20 (18:2:1 CHCl₃ :ethanol:acetic acid; the tlcplate was exposed to NH3 vapor prior to elution to convert the HCl saltto free base).

EXAMPLE 162R,4S,5S-6-Cyclohexyl-5-[N-(N-(t-butoxycarbonyl)phenylalanyl)norleucyl]amino-4-hydroxy-2-(2-methylpropyl)-N-methylhexanamide

Title product of the preceding Example (61.8 mg., 0.185 mmol), CH₂ Cl₂(0.5 ml.), triethylamine (0.033 ml., 0.240 mmol),[N-(t-butoxycarbonyl)phenylalanyl]norleucine (70 mg., 0.185 mmol),1-hydroxybenzotriazole (43 mg., 0.278 mmol) and dicyclohexylcarbodiimide(38 mg., 0.185 mmol) were added in sequence to a 2 ml. flask, themixture stirred 18 hours at 0°, and the mixture filtered. The filtratewas diluted with 2.5 ml. CH₂ Cl₂, washed 2×3 ml. 1N NaOH, 1×3 ml. brine,dried (MgSO₄) and stripped to an off white powder. The latter waschromatographed on 2 g. silica gel and gradiently eluted with 50 ml.each 0.5%, 1%, 2%, 4% and 6% ethanol in CH₂ Cl₂ to yield 21.1 mg. ofwhite powder contaminated with dicyclohexylurea (DCU). The latter wascombined with the product and DCU containing solids which were initiallyisolated from the reaction mixture and chromatographed on 15 g. silicagel gradiently eluted with 500 ml. each 1%, 2%, 3% and 4% ethanol in CH₂Cl₂. Product fractions, free of DCU, were combined and stripped to yieldtitle product, 60.4 mg.; ¹ H-nmr (DMSO-d₆) 300 mHz (ppm) includes0.9-0.95 m, 9H, C(CH₃)₂ and C--CH₃); 1.33 (s, 9H, boc); 2.56 (d, 3H,NCH₃, J=ca 5 Hz); 3.8, 4.18, and 4.32 (m, 1H, each); 4.62 (d, 1H, J=ca.4Hz); 7.06, 7.46 and 7.95 (d, 1H each, J=ca.8 Hz), 7.68 (q, 1H, J=ca 0.5Hz); 7.2-7.4 (m, aromatic).

EXAMPLE 172R,4S,5S-6-Cyclohexyl-5-(t-butoxycarbonylamino)-4-(2-tetrahydropyranyloxyl-2-(2-methylpropyl)-N-methylhexanamide

Title product of Example 8 (211 mg., 0.449 mmol) was dissolved in 1 ml.dry THF and cooled to -50° under N₂. With stirring, N-methylmorpholine(0.0545 ml., 0.494 mmol) was added and after 2-3 minutes,isobutylchloroformate (0.0641 ml., 0.494 mmol) was added dropwise. Afteran additional 10 minutes at -50°, the temperature was increased to -30°and methylamine (37.6 mg., 1.21 mmol) in 0.068 ml. of THF added dropwiseover 20 minutes. After 10 minutes, the mixture was warmed to ambienttemperature, diluted with 5 ml. of ethyl acetate, washed 3×5 ml.saturated NaHCO₃ and then 1×5 ml. H₂ O, dried (MgSO₄) and stripped to anoil, 245 mg. The latter was chromatographed on 5 g. silica gel elutingwith 1:1 ether:hexane and monitoring by tlc to yield title product as awhite solid foam, 102.2 mg.; tlc Rf 0.2 and 0.25 (3:1 ether:hexane),reflecting 1:1 epimers in the tetrahydropyranyl ether side chain.

EXAMPLE 182R,4S,5S-6-Cyclohexyl-5-(t-butoxycarbonylamino)-4-hydroxy-2-(2-methylpropyl)-N-methylhexanamide(Method B)

Title product of the preceding Example (98.6 mg., 0.204 mmol) wasdissolved in 1.5 ml. of 2:1 acetic acid:H₂ O and stirred for 6 hours,then stripped and chased 2×CH₃ C₆ H₅ to yield an oil, 99.2 mg. Thelatter was chromatographed on 5 g. silica gel eluting with 200 ml. 1:1,then 100 ml. 3:1 ethyl acetate: hexane, monitoring by tlc. Clean productfractions were combined and stripped to yield title product as a whitesolid foam, 61.5 mg.; tlc Rf 0.22 (2:1 ethyl acetate:hexane); ¹ H-nmrCDCl₃) 300 mHz (ppm) includes 0.9 (d, 6H), 1.46 (s, 9H), 2.82 (d, 3H);identical with title product of Example 14.

By the method of Example 15, the present product (59.2 mg.) wasconverted to2R,4S,5S-6-Cyclohexyl-5-amino-4-hydroxy-2-(2-methylpropyl)-N-methylhexaneamidehydrochloride, 50.0 mg., identical with the product of that Example.

EXAMPLE 192R,4S,5S-6-Cyclohexyl-5-[N-alpha-(N-t-butoxycarbonylphenylalanyl)-N(imidazole)-t-butoxycarbonyl)histidyl]amino-4-hydroxy-2-(2-methylpropyl)-N-methylhexanamide

By the method of Example 16, title product of Example 15 (47.3 mg.,0.141 mmol; prepared according to the immediately preceding Examples)andN-alpha-[N-(t-butoxycarbonyl)phenylalanyl]-N(imidazole)-(t-butoxycarbonyl)histidine(74.4 mg., 0.148 mmol) in 1 ml. CH₂ Cl₂ were converted to instant titleproduct. Following filtration of the reaction mixture, the filtrate wasstripped of CH₂ Cl₂ and diluted with 1 ml. ethyl acetate, washed 2×1 ml.1N NaOH, 1×1 ml. brine, dried (MgSO₄) and stripped to a foam 123.6 mg.,which was chromatographed on 4 g. silica gel, eluting with 100 ml. each0.5%, 1%, 2%, 4% and 6% ethanol in CH₂ Cl₂ and monitoring by tlc. Cleanproduct fractions were combined and stripped to yield title product as awhite solid foam, 84.4 mg.; tlc Rf 0.58 (18:2:1 CHCl₃ :ethanol aceticacid).

EXAMPLE 20 2R,4S,5S-6-Cyclohexyl-5-[N-alpha-(N-t-butoxycarbonylphenylalanyl)histidyl]amino-4-hydroxy-2-(2-methylpropyl)-N-methylhexanamide

Title product of the preceding Example (81.1 mg., 0.104 mmol) wasdissolved in 1 ml. methanol and stirred under N₂ with about 5 mg. of K₂CO₃ for 1 hour. The K₂ CO₃ was neutralized with acetic acid and themixture stripped to near dryness, diluted with water and desalted on ionexchange resin (5 g. RP C-18) eluting with 2×column volume of 2:3methanol:H₂ O and then 4×column volume of methanol. The non-aqueousfractions were combined, stripped and dried under high vacuum to yieldtitle product as a white powder, 63.5 mg.; tlc Rf 0.03 (18:2:1 CHCl₃:ethanol:acetic acid); ¹ H-nmr (DMSO-d₆) 300 mHz (ppm) includes: 0.90and 0.95 (doublets, 6H total, J=7 Hz, C(CH₃)₂); 1.30 s, 9H, C(CH₃)₃ ;2.56 (d, 3H, J=ca. 5 Hz, NCH₃); 3.72 (m, 1H); 4.14 (m, 1H); 4.47 (m,1H); 7.53 (s, 1H, one imidazolyl CH); 6.86, 7.12, 7.76 and 8.22 (m, 1Heach, NH); 7.16-7.40 (m, aromatic).

EXAMPLE 212-[2R,4S,5S-6-Cyclohexyl-5-(t-butoxycarbonylamino)-4-(2-tetrahydropyranyloxy)-2-(2-methylpropyl)hexanoyl]-1,2,3,4-tetrahydroisoquinoline

Title product of Example 8 (0.243 g., 0.517 mmol),tetrahydroisoquinoline (0.044 ml., 0.069 g., 0.517 mmol) and1-hydroxybenzotriazole (70 mg., 0.517 mmol) were combined with 5 ml. CH₂Cl₂ and cooled to 0°. Dicyclohexylcarbodiimide (107 mg., 0.517 mmol) wasadded and the mixture stirred 18 hours at room temperature, filtered,the filtrate stripped and the residue triturated with 5 ml. ethylacetate and refiltered. The second filtrate was washed 1×5 ml. 5% HCl,1×5 ml. saturated NaHCO₃ and 1×5 ml. brine, dried (MgSO₄), stripped andthe second residue chromatographed on 36 g. silica gel using 1% methanolin CHCl₃ as eluant, 229 mg.; ¹ H-nmr CDCl₃) includes delta 1.4 ppm. (s,9H, C(CH₂ Cl₃).

EXAMPLE 22 2-[2R,4S,5S-6-Cyclohexyl-5-amino-4-hydroxy-2-(2-methylpropyl)hexanoyl]-1,2,3,4-tetrahydroisoquinolineHydrochloride

By the method of Example 15, the product of the preceding Example (176mg., 0.301 mmol) was converted to incompletely dry, title product, 153mg.; ¹ H-nmr (CD₃ OD) includes delta 1.2 ppm (6H, J=7, --CH(CH₃)₂).

EXAMPLE 232-[2R,4S,5S-6-Cyclohexyl-5-(N-alpha-(N-t-butoxycarbonylphenylalanyl)-N(imidazole)-(t-butoxycarbonyl)histidyl)amino-4-hydroxy-2(2-methylpropyl)hexanoyl]-1,2,3,4-tetrahydroisoquinoline

By the method of Example 16, using N-methylmorpholine in place oftriethylamine, the wet product of the preceding Example (153 mg., 0.301mmol assumed) andN-alpha-(N-t-butoxycarbonylphenylalanyl)-N(imidazole)-(t-butoxycarbonyl)histidine(186 mg., 0.367 mmol) were converted to instant title product. Thereaction mixture was filtered and the filtrate chromatographed on silicagel using 2.5% methanol in CHCl₃ as eluant to yield present titleproduct, 286 mg.; ¹ H-nmr (CDCl₃) includes delta 1.4 ppm (s, 9H,C(CH₃)₃).

EXAMPLE 242-[2R.,4S,5S-6-Cyclohexyl-5-N-(N-t-butoxycarbonylphenylalanyl)histidyl)amino-4-hydroxy-2-(2-methylpropyl)hexanoyl]-1,2,3,4-tetrahydroisoquinoline

Title product of the preceding Example (286 mg.) was stirred for 6 dayswith 10 ml. 4:1 acetic acid:H₂ O, then stripped with toluene chase andthe residue chromatographed on 20 g. silica gel with 10% methanol inCHCl₃ as eluant to yield title product, the less polar of 2 products,110 mg.; ¹ H-nmr CDCl₃) includes 1.4 ppm (s, 9H, C(CH₃)₃).

EXAMPLE 25N-[2R,4S,5S-6-Cyclohexyl-5-(t-butoxycarbonylamino)-4-(2-tetrahydropyranyloxy)-2-(2-methylpropyl)hexanoyl]prolineMethyl Ester

Except to use a reaction time of 2 hours at 0° and 16 hours at roomtemperature, to use N-methylmorpholine in place of triethylamine, and touse 1% methanol in CHCl₃ as eluant on chromatography, the method ofExample 19 was employed to convert the product of Example 8 (300 mg.,0.639 mmol) and proline methyl ester hydrochloride (106 mg.; 0.639 mmol)to title produce, 215 mg.; ¹ H-nmr CDCl₃) includes delta 1.4 ppm (s, 9H,C(CH₃)₃); tlc Rf 0.7 (2.5% CH₃ OH in CHCl₃).

EXAMPLE 26N-[2R,4S,5S-6-Cyclohexyl-5-(t-butoxycarbonylamino)-4-hydroxy-2-(2-methylpropyl)hexanoyl]prolineMethyl Ester

Product of the preceding Example (165 mg.) was stirred in 10 ml. 4:1acetic acid:H₂ O for 18 hours, stripped, and chased 2×5 ml. toluene and1×5 ml. CHCl₃ to yield somewhat wet title product as an oil, 166 mg.;tlc Rf 0.6 (2.5% CH₃ OH in CHCl₃).

EXAMPLE 27N-[2R,4S,5S-6-Cyclohexyl-5-amino-4-hydroxy-2-(2-methylpropyl)hexanoyl]prolineMethyl Ester

Title product of the preceding Example (166 mg., wet) was combined with3.7N HCl in dioxane (1 ml.). After 2 hours, the mixture was stripped,taken up in 5 ml. ethyl acetate, washed 1×5 ml. saturated NaHCO₃ andthen 1×5 ml. brine, dried (MgSO₄) and restripped to yield title product,¹ H-nmr (CD₃ OD) includes delta 3.8 ppm (s, 3H, CO₂ CH₃).

EXAMPLE 28N-[2R,4S,5S-6-Cyclohexyl-5-(N-alpha-(N-t-butoxycarbonylphenylalanyl)-N(imidazole)-(t-butoxycarbonyl)histidyl)amino-4-hydroxy-2-(2-methylpropyl)hexanoyl]prolineMethyl Ester

The product of the preceding Example (132 mg., 0.334 mmol),N-alpha-(N-t-butoxycarbonylphenylalanyl)-N-(imidazole)-(t-butoxycarbonyl)histidine(168 mg., 0.334 mmol), 1-hydroxybenzotriazole (45 mg., 0.334 mmol) anddicyclohexylcarbodiimide (69 mg., 0.334 mol) were combined in 5 ml. CH₂Cl₂ at 0°, protected by a CaCl₂ drying tube, and stirred 18 hours atthat temperature, then stripped of solvent, the residue triturated with5 ml. ether and filtered. The ether filtrate was stripped to a residue(230 mg.) which was chromatographed on silica gel with 1% CH₃ OH inCHCl₃ as eluant to yield purified title product, 110 mg.; ¹ H-nmr(CDCl₃) includes delta 1.4 ppm (s, 9H, C(CH₃)₃).

EXAMPLE 292R,4S,5S-6-Cyclohexyl-5-[N-alpha-(N-t-butoxycarbonylphenylalanyl)histidyl]amino-2-(2-methylpropyl)-gamma-hexanolactone

By the method of Example 26, title product of the preceding Example wasconverted toN-[2R,4S,5S-6-Cyclohexyl-5-(N-alpha-(N-t-butoxycarbonylphenylalanyl)-N(imidazole)-(t-butoxycarbonyl)histidyl)amino-4-hydroxy-2-(2-methylpropyl)hexanoyl]prolinemethyl ester, 110 mg.; ¹ H-nmr showed loss of the histidinet-butoxycarbonyl protecting group. This intermediate product, whenchromatographed on silica gel with 1:24 CH₃ OH:CHCl₃ formed titleproduct with loss of proline methyl ester, 51 mg.; ¹ H-nmr (CDCl₃)includes delta 1.4 ppm (s, 9H, C(CH₃)₃), but no methyl ester peak; ir(CDCl₃) includes lactone carbonyl at 1785 cm⁻¹.

EXAMPLE 302R,4S,5S-6-Cyclohexyl-5-[N-alpha-(N-t-butoxycarbonylphenylalanyl)histidyl]amino-4-hydroxy-2-(2-methylpropyl)hexanamide

Title product of the preceding Example (32.7 mg.) on 0.5 ml. methanolwas perfused with excess dry NH₃ at 0°. After about 10 minutes, thereaction mixture was stripped, the residue triturated with ether, andtitle product recovered as a white solid by filtration, 23 mg.; ¹ H-nmrCDCl₃) includes delta 1 4 ppm (s, 9H, C(CH₃)₃).

EXAMPLE 31N-[N-alpha-(t-butoxycarbonyl)-N-epsilon-(benzyloxycarbonyl)lysyl]statineEthyl Ester

By the method of Example 19, chromatographic purification beingunnecessary, statine ethyl ester hydrochloride (0.467 g., 0.00195 mol),N-alpha-(t-butoxycarbonyl)-N-epsilon-(benzyloxycarbonyl)lysine 0.198 g.,0.00195 mol), N-methylmorpholine (0.215 ml., 198 mg., 0.00195 mol),1-hydroxybenzotriazole (264 mg., 0.00195 mol) anddicyclohexylcarbodiimide (403 mg., 0.00195 mol) in 25 ml. CH₂ Cl₂ wereconverted to title product as a solid foam, 0.704 g.; tlc Rf 0.65 (9:1CHCl₃ : CH₃ OH); ¹ H-nmr CDCl₃) includes delta 1.4 ppm (s, 9H, C(CH₃)₃).

EXAMPLE 32 N-[N-epsilon-(Benzyloxycarbonyl)lysyl]statine Ethyl EsterHydrochloride

The product of the preceding Example (0.805 g., 0.00142 mol) was stirredin 10 ml. 3.7N HCl in dioxane for 2 hours, stripped, the residuetriturated with ether, and filtered to yield title product, 0.650 g.; ¹H-nmr (CD₃ OD) includes delta 5.2 ppm (s, 2H, benzyl-CH₂).

EXAMPLE 33N-[N-alpha-(2R,4S,5S-6-Cyclohexyl-5-(t-butoxycarbonylamino)-4-(2-tetrahydropyranyloxy)-2-(2-methylpropyl)hexanoyl)-N-epsilon-(benzyloxycarbonyl)lysyl]statineEthyl Ester

By the procedure of Example 28, using 0.5% methanol in CHCl₃ as eluant,the product of the preceding Example (332 mg., 0.662 mmol) and theproduct of Example 8 (311 mg., 0.662 mmol) were converted tochromatographed title product, 300 mg.; ¹ H-nmr (CDCl₃) includes delta1.4 (s, 9H, C(CH₃)₃).

EXAMPLE 34N-[N-alpha-(2R,4S,5S-6-Cyclohexyl-5-(t-butoxycarbonylamino)-4-hydroxy-2-(2-methylpropyl)-hexanoyl)-N-epsilon-(benzyloxycarbonyl)lysyl]statineEthyl Ester

By the procedure of Example 26, the product of the preceding Example(300 mg.) was converted to title product, chased with 2×10 ml. oftoluene and dried under high vacuum, 200 m9.: ¹ H-nmr (CDCl₃) includesdelta 1.4 ppm (s, 9H, C(CH₃)₃).

EXAMPLE 35N-N-alpha-[2R,4S,5S-6-Cyclohexyl-5-amino-4-hydroxy-2-(2-methylpropyl)hexanoyl)-N-epsilon(benzyloxycarbonyl)lysyl]statineEthyl Ester

The product of the preceding Example (200 mg.) and 10 ml. of 3.7N HCl indioxane were stirred for 2 hours, stripped, the residue triturated withether and white solids recovered by filtration. Chromatography of thesesolids on 35 g. silica gel using 1:19 CH₃ OH:CHCl₃ gave HCl free titleproduct, 52 mg.; ¹ H-nmr (CD₃ OD) includes delta 5.2 (s, 2H,benzyl-CH₂).

EXAMPLE 36N-[[N-alpha-[2R.,4S,5S-6-Cyclohexyl-5-((N-alpha-(N-t-butoxycarbonyl:phenylalanyl)-N(imidazole)-(t-butoxycarbonyl)histidyl))amino-4-hydroxy-2(2-methylpropyl)hexanoyl]-N-epsilon-[benzyloxycarbonyl]lysyl]]statineEthyl Ester

Except to use 2 equivalents of 1-hydroxybenzotriazole and to use 2.5%CH₃ OH in CHCl₃ as eluant, the product of the preceding Example (52 mg.,0.0709 mmol) andN-alpha-(N-t-butoxycarbonylphenylalanyl)-N(imidazole)-(t-butoxycarbonyl)histidine(36 mg., 0.0709 mol) were converted to chromatographed, title product,28 mg.; ¹ H-nmr CDCl₃) includes delta 1.4 ppm (s, 9H, C(CH₃)₃); hplcretention time 10.28 minutes (reverse phase C18 column eluting with 1:1acetonitrile:pH 2.1 phosphate buffer at 3 ml/minute).

EXAMPLE 37N-[[N-alpha-2R,4S,5S-6-Cyclohexyl-5-(N-butoxycarbonylphenylalanyl-histidyl)amino-4-hydroxy-2-(2-methylpropyl)hexanoyl]-N-epsilon-[benzyloxycarbonyl]lysyl]]statineEthyl Ester

By the method of Example 26, the product of the preceding Example (28mg.) was converted to present title product, 22 mg.; ¹ H-nmr (CDC13)demonstrated loss of the imidazole t-butoxycarbonyl protecting group,but includes delta 1.4 ppm (s, 9H) due to the retained t-butoxycarbonylgroup.

EXAMPLE 38N-[[N-alpha-2R.,4S,5S-6-Cyclohexyl-5-(N-t-butoxycarbonylphenylalanyl-histidyl)amino-4-hydroxy-2-2-methylpropyl)hexanoyl]lysyl]]statineEthyl Ester

The product of the preceding Example (22 mg.) was hydrogenated atatmospheric pressure in 2 ml. ethanol over 22 mg. 10% Pd/C for 3 hours.Catalyst was recovered by filtration over diatomaceous earth with 2×1ml. ethanol wash. The combined filtrate and wash was stripped to aglass, converted to a white, filterable powder by trituration withminimal ether, 13 mg.; ¹ H-nmr CDCl₃) includes delta 1.5 ppm (s, 9H,C(CH₃)₃).

EXAMPLE 392R,4S,5S-6-Cyclohexyl-5-(t-butoxycarbonylamino)-2-benzyl-gamma-hexanolactone

Under N₂, dry di(isopropyl)amine (2.48 ml., 0.0177 mol; distilled fromCaH₂) in dry THF (7.4 ml., distilled from K) was cooled to 0°.Butyllithium (11.0 ml. of 1.62M in hexane, 0.0177 mol) was addeddropwise over 5 minutes. After stirring 15 minutes at 0°, the mixturewas cooled to -78° and the less polar 4S,5S-title product of Example 3(2.30 g., 0.0074 mol) in 3.7 ml. dry THF added over 5 minutes. After

stirring 30 minutes more at -78°, benzyl bromide (0.923 ml., 0.0078 mol)in 3.7 ml. dry THF was added dropwise over 5 minutes. After 1 hour at-78°, the reaction was quenched by the addition of 10 ml. saturated NH₄Cl, warmed to room temperature, diluted with 25 ml. ether and the layersseparated. The organic layer was washed 2×15 ml. saturated NaHCO₃, dried(MgSO₄) and stripped to an oil (3.29 g.). The oil was chromatographed on150 g. silica gel eluting with 2.5 liters 1:9 ethyl acetate:hexane andmonitoring by tlc. Clean product fractions were combined and stripped toyield purified title product as a white oily solid, 1.46 g.; tlc Rf 0.60(1:1 ethyl acetate:hexane). More polar starting material (0.91 g. ofyellow oil about 70% pure, tlc Rf 0.4 in the same system) was alsorecovered from the column.

EXAMPLE 402R,4S.,5S-6-Cyclohexyl-5-t-butoxycarbonylamino-4-hydroxy-2-benzyl-N-methylhexanamide

Title product of the preceding Example (157 mg.) was dissolved in 2 ml.CH₃ OH, cooled to 0°, and perfused with CH₃ NH₂ for 3 minutes. The flaskwas stoppered, allowed to stand at room temperature for 1.5 hours,stripped with 2×2 ml. ether chase and dried under high vacuum to yieldtitle product as a white, solid foam, 164 mg.; tlc Rf 0.17 (1:1 ethylacetate:hexane), Rf 0.29 (3:1 ethyl acetate:hexane); ¹ H-nmr (CDCl₃, 300MHz showed the expected C(CH₃)₃ (s, 9H), N-CH₂ Cl₂ (d, 3H), aromaticresonances and t-butoxycarbonyl-NH (d, 1H), peaks.

EXAMPLE 412R,4S,5S-6-Cyclohexyl-5-amino-4-hydroxy-2benzyl-N-methylhexanamideHydrochloride

The product of the preceding Example (159 mg.) was dissolved in 2 ml. 4NHCl in dioxane, stirred under N₂ 0.5 hour, stripped, chased 3×2 ml.ether and the residue dried under high vacuum to yield title product asa pale yellow powder, 135 mg.; tlc 0.20 (18:2:1 CHCl₃ :ethanol: aceticacid; spotted plate exposed to NH₃ to neutralize HCl salt prior toelution).

EXAMPLE 422R,4S,5S-6-Cyclohexyl-5-[N-alpha-(N-t-butoxycarbonylphenylalanyl)-N(imidazole)-(t-butoxycarbonyl)histidyl]amino-4-hydroxy-2-benzyl-N-methylhexanamide

Title product of the preceding Example (123 mg., 0.333 mmol), CH₂ Cl₂ (1ml.), triethylamine (0.060 ml., 0.433 mmol),N-alpha-N-t-butoxycarbonylphenylalanyl)-N(imidazole)-(t-butoxycarbonyl)histidine(176 mg., 0.350 mmol), 1-hydroxybenzotriazole (84 mg., 0.550 mmol) anddicyclohexylcarbodiimide (72 mg., 0.350 mmol) were sequentially combinedat 0° and stirred under N₂ for 16 hours at that temperature. By-productDCU was recovered by filtration with 3 ml. CH₂ Cl₂ wash. The combinedfiltrate and wash was washed 2×3 ml. 1N NaOH, and then 1×3 ml. brine,dried (MgSO₄), stripped to 342 mg. pale white solids, andchromatographed on 20 g. silica gel, gradiently eluting with 250 ml.each 0.5%, 1%, 2%, 4% and 6% ethanol in CH₂ Cl₂ and monitoring by tlc toyield purified title product, 154 mg.; tlc Rf 0.55 (18:2:1 CHCl₃ethanol:acetic acid).

EXAMPLE 432R,4S,5S-6-Cyclohexyl-5-(t-butoxycarbonylphenylalanyl-histidyl)amino-4-hydroxy-2-benzyl-N-methylhexanamide

Title product of the preceding Example (147 mg.) was stirred under N₂with 1.5 ml. of 4:1 acetic acid:H₂ O for 7 hours, then stripped with 3×3ml. ether chase and dried under high vacuum to yield title product as awhite powder, 135 mg.; tlc Rf 0.05 (18:2:1 CH₃ Cl₃ :ethanol:aceticacid); ¹ H-nmr (DMSO-d₆), 250 MHz includes delta (ppm): 1.32 (s, 9H,t-butyl); 3.72, 4.15 and 4.48 (3m, 1H, each); 6.87 and 7.52 (s, 1H,each, imidazolyl CH); 7.0-7.35 (m, aromatic), 7.40, 7.62 and 8.17 (3d,1H each).

EXAMPLE 442R,4S,5S-6-Cyclohexyl-5-(t-butoxycarbonylamino-4-hydroxy-2-(cyclohexylmethyl)-N-methylhexanamide

Title product of Example 40 (83.1 mg.) was hydrogenated over 45 mg. of10% Rh/C in 10 ml. methanol under 4 atmospheres of H₂ for 1.5 hours.Catalyst was recovered by filtration over diatomaceous earth withmethanol wash. The combined filtrate and wash was stripped, chased 2×5ml. ether and dried in high vacuum to yield title product as a white,solid foam, 76.9 mg.; ¹ H-nmr (CDCl₃, 300 MHz) includes C(CH₃)₃ singlet,no phenyl protons, and N-CH₃ doublet; tlc Rf 0.32 (3:1 ethylacetate:hexane).

EXAMPLE 452R,4S,5S-6-Cyclohexyl-5-amino-4-hydroxy-2-(cyclohexylmethyl)-N-methylhexanamideHydrochloride

By the method of Example 41, title product of the preceding Example (75mg.) was converted to present title product, 64.1 mg.; tlc Rf 0.18(18:2:1 CHCl₃ :ethanol:acetic acid; spotted plate neutralized with NH₃vapor before elution).

EXAMPLE 462R,4S,5S-6-Cyclohexyl-5-[N-alpha-(N-t-butoxycarbonylphenylalanyl)-N(imidazole)-(t-butoxycarbonyl)histidyl]amino-4-hydroxy-2-(cyclohexylmethyl)-N-methylhexanamide

By the method of Example 42, the product of the preceding Example (59.1mg., 0.158 mmol) was converted to chromatographed title product as awhite, solid foam, 76.4 mg.; tlc Rf 0.60 (18:2:1 CHCl₃ :ethanol:aceticacid); ¹ H-nmr (DMSO-d₆, 250 MHz) includes two C(CH₃)₃ singlets centeredat 1.6 ppm.

EXAMPLE 472R,4S,5S-6-Cyclohexyl-5-(t-butoxycarbonylphenylalanyl-histidyl)amino-4-hydroxy-2-(cyclohexylmethyl)-N-methylhexanamide

By the method of Example 43, the product of the preceding Example (71.5mg.) was converted to title product as a white powder, 59.1 mg.; tlc Rf0.05 (18:2:1 CHCl₃, ethanol:acetic acid), ¹ H-nmr (DMSO-d₆, 250 MHz)includes C(CH₃)₃ singlet at 1.32 ppm, N-CH₃ doublet at 2.58 ppm andimidazole C-H singlets at 6.86 and 7.52 ppm.

EXAMPLE 482R,4S,5S-6-Cyclohexyl-5-(t-butoxycarbonylamino)-4-hydroxy-2-(2-methylpropyl)-N-benzylhexanamide

Title product of Example 5 (150 mg., 0.408 mmol), dry toluene (2 ml.),benzylamine (0.29 ml., 5 equivalents), acetic acid (0.023 ml., 1equivalent) were stirred under N₂, gradually heated to 90° and held atthat temperature for 6 hours. The reaction mixture was cooled, dilutedwith 3 ml. ethyl acetate, washed 2×3 ml. 1N HCl and 1×3 ml. brine, dried(MgSO₄) and stripped to yield title product as a white, oily foam, 187mg.; tlc Rf 0.58 (2:1 ethyl acetate:hexane).

EXAMPLE 492R,4S,5S-6-Cyclohexyl-5-amino-4-hydroxy-2-(2-methylpropyl)-N-benzylhexanamideHydrochloride

By the method of Example 41, the product of the preceding Example (172mg.) was converted to present title product as a white powder, 146 mg.;tlc Rf 0.30 (18:2:1 CHCl₃ :ethanol:acetic acid; spotted plate exposed toNH₃ vapor prior to elution).

EXAMPLE 502R,4S,5S-6-Cyclohexyl-5-[N-alpha-(N-t-butoxycarbonylphenylalanyl)-N(imidazole)-(t-butoxycarbonyl)histidyl]amino-4-hydroxy-2-(2-methylpropyl)-N-benzylhexanamide

By the method of Example 42, the product of the preceding Example (136mg., 0.331 mmol) was converted to chromatographed title product as awhite, solid foam, 167 mg.; tlc Rf 0.60 (18:2:1 CHCl₃ :ethanol:aceticacid).

EXAMPLE 512R,4S,5S-6-Cyclohexyl-5-(t-butoxycarbonylphenylalanyl-histidyl)amino-4-hydroxy-2-(2-methylpropyl)-N-benzylhexanamide

By the method of Example 43, the product of the preceding Example wasconverted to present title product as a white, solid foam, 146 mg.; tlcRf 0.18 (18:2:1 CHCl₃ :ethanol:acetic acid); ¹ H-nmr (DMSO-d₆), 250 MHz)includes delta (ppm): 0.83 and 0.90 (d, 3H, each, J=6 Hz, C(CH₃)₂); 1.32(s, 9H, C(CH₃)₃); 3.72, 4.16 and 4.50 (m 1H, each); 4.28 (m, 2H, NCH₂);6.86 (br, 1H), 7.52 (s, 1H); 7.1-7.4 (m, 11-13H, aromatic and NH); 8.25(d, 1H, NH); 8.36 (t, 1H).

EXAMPLE 522R,4S,5S-6-Cyclohexyl-5-(t-butoxycarbonylamino)-4-hydroxy-2-(2-methylpropyl)-N,N-dimethylhexanamide

Title product of Example 5 (595 mg.) was dissolved in 5 ml. methanol,chilled to 10°, perfused with dimethylamine for 3 minutes, stoppered andallowed to stand at room temperature for 1.5 hours. Tlc indicated 80%conversion. At room temperature the solution was then perfused for 2minutes with dimethylamine, restoppered, and allowed to stand 18 hours,at which time tlc indicated complete conversion. The solution wasstripped and the residue dried under high vacuum to yield title productas a white, solid foam, 112 mg.; tlc Rf 0.31 (3:1 ethyl acetate:hexane);ms includes peaks at 281.0, 230.9, 212.0, 186.1, 131.0, 100.0, 80.9,68.9 and 57.0.

EXAMPLE 532R,4S,5S-6-Cyclohexyl-5-amino-4-hydroxy-2-(2-methylpropyl)-N,N-dimethylhexanamideHydrochloride

By the method of Example 41, the product of the preceding Example (107mg.) was converted to present title product as an off-white, solid foam,94.7 mg.; tlc Rf 0.16 (18:2:1 CHCl₃ :ethanol:acetic acid; spotted plateexposed to NH₃ vapor prior to elution); ms includes 313.3, 256.1, 215.2,186.1, 156.0, 143.1, 126.1, 111.1, 100.1, 83.0 and 69.1.

EXAMPLE 542R,4S,5S-6-Cyclohexyl-5-[N-alpha-(N-t-butoxycarbonylphenylalanyl)-N(imidazole)-(t-butoxycarbonyl)histidyl]amino-4-hydroxy-2-(2-methylpropyl)-N,N-dimethylhexanamide

By the method of Example 42, additionally using 250 ml. each of 8%, 12%,20% and 50% ethanol in CH for chromatographic elution, the product ofthe preceding Example (87 mg., 0.249 mmol) was converted to presenttitle product as a white, solid foam, 107 mg.; tlc Rf 0.52 (18:2:1 CHCl₃:ethanol:acetic acid); ¹ H-nmr (DMSO-d₆, 250 MHz) include two C(CH₃)₃singlets, aromatic peaks and imidazole peaks; ms 511.4, 357.3, 329.1,301.2, 257.0, 211.1, 165.1, 136.0, 120.1, 110.0, 91.0, 57.1, 41.0.

EXAMPLE 552R,4S,5S-6-Cyclohexyl-5-(t-butoxycarbonylphenylalanyl-histidyl)amino-4-hydroxy-2-(2-methylpropyl)-N,N-dimethylhexanamide

By the method of Example 43, the product of the product of the precedingExample was converted to crude title product, 78.2 mg., containing asubstantial mp impurity by hplc. The crude product was subjected topreparative hplc on a 5 g. reverse phase C-18 column, eluted with fourcolumn volumes each of 1:1, 3:1, 17:3 and 100:0 methanol:H₂ O. Theimpurity eluted with the 3:1 eluant, while purified title product elutedwith the 17:3 eluant, isolated by stripping as a white powder, 46.0 mg.;tlc Rf 0.02 (18:2:1 CHCl₃ :ethanol:acetic acid); hplc retention time5.87 minutes (C-8 4.6 mm×25 cm. column, eluted with 3:2 CH₃ CN:pH 2.1phosphate buffer at 1.5 ml/min.); ms 322.4, 311.1, 283.1, 218.9, 165.1,131.0, 110.1, 97.0, 91 0, 69.1, 59.1, 43.0; ¹ H-nmr (DMSO-d₆, 250 MHz)includes delta 1.32 (s, 9H, C(CH₃)₃, 2.85 and 3.07 (2 s, 6H, N(CH₃)₂),6.92 and 7.52 (2s, 2H, imidazole C--H).

EXAMPLE 565-[2R,4S,5S-6-Cyclohexyl-5-(t-butoxycarbonylamino)-4-(2-tetrahydropyranyloxy)-2-(2-methylpropyl)hexanoyl]amino-1-(benzyloxycarbonylamino)-pentane

By the method of Example 31, except to use ether in place of ethylacetate in isolation, title product of Example 8 (160 mg., 0.341 mmol)and of Preparation 3 (80.5 mg., 0.341 mmol) were converted to presenttitle product, 222 mg.; ¹ H-nmr (CDCl₃) includes delta 1.4 ppm (s, 9H,C(CH₃)₃).

EXAMPLE 575-[2R,4S,5S-6-Cyclohexyl-5-(t-butoxyamino)-4-hydroxy-2-(2-methylpropyl)hexanoyl]amino-1-(benzylcarbonylamino)pentane

By the method of Example 26, the product of the preceding Example (221mg.) was converted to present title product, 193 mg.; ¹ H-nmr (CDCl₃)includes delta 1.4 ppm (s, 9H, C(CH₃)₃).

EXAMPLE 585-[2R,4S,5S-6-Cyclohexyl-5-amino-4-hydroxy-2-(2-methylpropyl)hexanoyl]amino-1-(benzyloxycarbonylamino)pentane

The product of the preceding Example (193 mg.) was stirred with 10 ml.3.7N HCl in dioxane for 2 hours, stripped and the residue trituratedwith ether to yield title product, 144 mg.; ¹ H-nmr (CD₃ OD) includesdelta 5.2 ppm (s, 2H, benzyl CH₂).

EXAMPLE 595-[[2R,4S,5S-6-Cyclohexyl-5-[N-alpha-(N-t-butoxycarbonylphenylalanyl)-N(imidazole)-(t-butoxycarbonyl)histidyl]amino-4-hydroxy-2-(2-methylpropyl)hexanoyl]]amino-1-(benzylcarbonylamino)pentane

Using CHCl₃ as eluant, the method of Example 28 was employed to convertthe product of the preceding Example to present chromatographed titleproduct, 25 mg.; ¹ H-nmr (CDCl₃) includes delta 1.4 ppm (s, 9H, C(CH₃)₃.

EXAMPLE 605-[2R,4S,5S-6-Cyclohexyl-5-(t-butoxycarbonylphenylalanyl-histidyl)amino-4-hydroxy-2-(2-methylpropyl)hexanoyl]amino-1-(benzyloxycarbonylamino)pentane

By the method of Example 43, title product of the preceding Example (25mg.) was converted to present title product, the residue after strippingwas chased twice with toluene and the residue triturated with ether toyield title product as white solids, 11 mg.; ¹ H-nmr (CDCl₃) includesdelta 1.4 ppm (s 9H, C(CH₃)₃).

Hydrogenation over Pd/C according to Example 13 is used to convert thisproduct to5-[2R,4S,5S-6-Cyclohexyl-5-(t-butoxycarbonylphenylalanyl-histidyl)amino-4-hydroxy-2-(2-methylpropyl)hexanoyl]aminopentylamine.

EXAMPLE 61Benzyl4-[2R,4S,5S-6-Cyclohexyl-5-(t-butoxycarbonylamino)-4-(2-tetrahydropyranyloxy)-2-(2-methylpropyl)hexanoyl]aminobutyrate

Title product of Example 8 (160 mg., 0.341 mmol) and benzyl4-aminobutyrate hydrochloride (66 mg., 0.341 mmol) were coupledaccording to the procedure of Example 23. At the end of the reactionperiod, the mixture was stripped and the residue taken up in 5 ml.ether, washed 1×3 ml. saturated NaHC₃, 1×3 ml. 5% HCl and 1×3 ml. brine,and restripped to crude product, 172 mg., which was chromatographed on20 g. silica gel with CHCl₃ as eluant to yield purified title product,107 mg.; ¹ H-nmr (CDCl₃) includes delta 1.4 ppm (s, 9H, C(CH₃)₃).

EXAMPLE 62 Benzyl4-[2R,4S,5S-6-Cyclohexyl-5-(t-butoxycarbonylamino)-4-hydroxy-2-(2-methylpropyl)-hexanoyl]aminobutyrate

By the method of Example 43, the product of the preceding Example (380mg.) was converted to present title product, 330 mg.; ¹ H-nmr includesdelta 1.4 ppm (s, 9H, C(CH₃)₃).

EXAMPLE 63 Benzyl4-[2R,4S,5S-6-Cyclohexyl-5-amino-4-hydroxy-2-(2-methylpropyl)hexanoyl]aminobutyrate

The product of the preceding Example (330 mg.) in 10 ml. of 3.7N HCl indioxane was allowed to stand for 2 hours. The mixture was stripped, theresidue was taken up in 5 ml. ethyl acetate, washed with 5 ml. each ofsaturated NaHCO₃ and then brine, dried (MgSO₄) and restripped to yieldtitle product as an oil, 220 mg.; ¹ H-nmr (CD₃ OD) includes delta 5.2ppm (s, 2H, CH₂ C₆ H₅).

EXAMPLE 64 Benzyl4-[[2R,4S,5S-6-Cyclohexyl-5-[N-alpha-(N-(t-butoxycarbonyl)phenylalanyl)-N(imidazole)-(t-butoxycarbonyl)histidyl]amino-4-hydroxy-2-(2-methylpropyl)hexanoylamino]butyrate

By the method of Example 42, the product of the preceding Example (270mg., 0.586 mmol) was coupled to form title product. Following recoveryof DCU, the filtrate was stripped, taken up in ether, refiltered, andthe filtrate stripped to 480 mg. of crude product. The latter waschromatographed on 50 g. silica gel using 2.5% methanol in CHCl₃ aseluant to yield purified title product, 132 mg.; ¹ H-nmr (CDCl₃)includes delta 1.4 ppm (s, 9H, C(CH₃)₃).

EXAMPLE 654-[2R,4S,5S-6-Cyclohexyl-5-(N-(t-butoxycarbonyl)phenylalanyl-histidyl)amino-4-hydroxy-2-(2-methylpropyl)hexanoylamino]butyricAcid

The product of the preceding Example (93 mg.) was hydrogenated in 2.5ml. of 1:1 ethyl acetate:methanol over 23 mg. of 10% Pd/C for 2 hours at4 atmospheres. Catalyst was recovered by filtration and the filtratestripped to an oil, which was taken up in ether, filtered and restrippedto yield title product, 62 mg.; ¹ H-nmr (CD₃ OD) includes delta 1.4 ppm(s, 9H, C(CH₃)₃).

EXAMPLE 662R,4S,5S-6-Cyclohexyl-5-(t-butyloxycarbonylamino)-2-[(2-naphthyl)methyl]-gamma-hexanolactone

By the method of Example 39, title product of Example 3 (0.295 g., 0.95mmol) and 2-(bromomethyl)-naphthalene (220 mg., 0.99 mmol) wereconverted to crude title product (0.43 g. of yellow oil) which waschromatographed on 20 g. silica gel, eluting with 700 ml. 1:9 ethylacetate:hexane and then 300 ml. 1:1 ethyl acetate:hexane, monitoring bytlc. Clean title product was isolated from center cuts, 139 mg.; Rf 0.62(1:1 ethyl acetate:hexane).

EXAMPLE 672R,4S,5S-6-Cyclohexyl-5-(t-butoxycarbonylamino)-4-hydroxy-2-[(2-naphthyl)methyl]-N-methylhexanamide

Title product of the preceding Example (113 mg.) in 2 ml. of methanolwas chilled to 0°-5° and the cold solution perfused with CH₃ NH₂ for 2minutes, stoppered, allowed to stand at ambient temperature for 2 hours,stripped, chased 2×2 ml. ether and dried under high vacuum to yieldtitle product as a white foam, 114 mg.; tlc Rf 0.15 (1:1 ethylacetate:hexane), Rf 0.48 (18:2:1 CHCl₃ :ethanol:acetic acid).

EXAMPLE 682R,4S,5S-6-Cyclohexyl-5-amino-4-hydroxy-2-[(2-naphthyl)methyl]-N-methylhexanamideHydrochloride

By the method of Example 15, costripping with ether, the product of thepreceding Example 107.4 mg., was converted to present title product, asolvent wet yellow powder, 102.5 mg., Rf 0.2 (18:2:1 CHCl₃:ethanol:acetic acid; loaded plate preneutralized with NH₃ vapor).

EXAMPLE 692R,4S,5S-6-Cyclohexyl-5-(N-N-alpha-(N-t-butoxycarbonyl)phenylalanyl)-N(imidazole)-(t-butoxycarbonyl)histidyl]amino-4-hydroxy-2-[(2-naphthyl)methyl]-N-methylhexanamide

By the method of Example 16, title product of the preceding Example (96mg. 0.229 mmol) and title product of Preparation 1 (121 mg., 0.240 mmol)were converted to crude title product as an oily foam, 235 mg., whichwas chromatographed on 5 g. silica gel, eluting with 200 ml. each of0.5%, 1%, 2%, 4% and 6% ethanol in CH₂ Cl₂ and monitoring by tlc. Cleanproduct fractions were stripped to yield purified title product a awhite foam, 124 mg., tlc Rf 0.6 (18:2:1 CHCl₃ :ethanol:acetic acid).

EXAMPLE 702R,4S,5S-Cyclohexyl-5-(N-t-butoxycarbonylphenyl-alanyl-histidyl)amino-4-hydroxy-2-[(2-naphthyl)-methyl)-N-methylhexanamide

Title product of the preceding Example (116 mg.) in 1.5 ml. methanol wasstirred with a K₂ CO₃ (1 mg.) for 1 hour, then neutralized with 2 dropsof acetic acid. The mixture was costripped with ether to yield titleproduct as a white powder, 101.4 mg.; tlc Rf 0.08 (18:2:1:ethanol:aceticacid); ms no m⁺ but includes mass peaks 311.1, 283.0, 257.0, 213.0,192.1, 179.1, 141.0, 111.0, 97.1 and 57.1; 1H-nmr (DMSO-d₆) delta (300MHz), 1.30 (s, 9H, C(CH₃)₃), 2.46 (d, 3H, NCH₃), 3.70 4.14, 4.47 (3m, 1Heach), 6.86, 7.62 (2s, 1H each), 7.10, 8.22 (2d, 1H each), 7.15-7.41 (m,aromatic), 7.48, 7.84 (2m, aromatic).

EXAMPLE 712R,4S,5S-6-Cyclohexyl-5-[[N-[N-alpha-methyl-N-alpha-(N-t-butoxycarbonylphenylalanyl)-N(imidazole)-(t-butoxycarbonyl)]histityl]]amino-4-hydroxy-2-(2-(2-methylpropyl)-N,N-dimethylhexanamide

By the methods of Examples 16 and 69, the product of Example 53 (0.534g., 0.00153 mol) andN-alphamethyl-N-alpha-(N-t-butoxycarbonylphenylalanyl)-N(imidazole)-(t-butoxycarbonyl)histidine(0.790 g., 0.00153 mol) were converted to present, chromatographedproduct as an off-white foam, 0.394 g. tlc Rf 0.52(18:2:1:ethanol:acetic acid).

EXAMPLE 722R,4S,5S-6-Cyclohexyl-5-[N-alpha-methyl-N-alpha-(N-(t-butoxycarbonyl)phenylalanyl)histidyl]amino-4-hydroxy-2-(2-methylpropyl)-N,N-dimethylhexanamide

By the method of Example 70, the product of the preceding Example (195mg.) was converted to present title product, purified by preparativehplc on an RP C-18 column, eluting with 4 column volumes of 1:1 CH₃OH:H₂ O and 6 column volumes of 3:1 CH₃ OH:H₂ O. Clean product fractionswere found in the latter eluant, recovered as a white powder bystripping, 153.6 mg.; tlc Rf 0.09 (18:2:1 CHCl₃ :ethanol:acetic acid);ms no m⁺ but includes 575.3, 525.3, 511.3, 472.3, 442.2, 416.2, 399.2,288.2, 371.2, 343.1, 291.0, 247.1, 186.1, 165.1, 123.9, 109.0, 95.0,69.1, 59.0, 43.0; ¹ H-nmr (DMSO-d₆) delta (300 MHz) 0.78 (2d, 6H (CH₃)₂rotamers), 3.0 (3s, 3H), 3.10, 3.76, 4.50, 4.60, 4.95, 5.10, 6.92, 7.48,7.60 (multiplets), 7.10-7.35 (broad multiplets, aromatic).

EXAMPLE 73N-alpha-[2R,4S-5S-6-Cyclohexyl-5-(t-butoxycarbonyl)amrno-4-(2-tetrahydropyranyloxy)-2-(2-methylpropyl)hexanoyl]-N-epsilon-[benzyloxycarbonyl]lysineMethyl Ester

Title product of Example 8 (0.318 g., 0.00068 mol) was coupled withN-epsilon-(benzyloxycarbonyl)lysine methyl ester hydrochloride (0.223g., 0.00068 mol) according to the method of Example 9. The crude product(0.261 g.) was purified by chromatography on 30 g. silica gel with 99:1CHCl₃ :CH₃ OH as eluant to yield purified title product, 0.147 g.; ¹H-nmr (CDCl₃) includes delta 3.8 (s, 3H, OCH₃).

EXAMPLE 74N-alpha-[2R,4S,5S-6-Cyclohexyl-5-(t-butoxycarbonyl)-amino-4-hydroxy-2-(2-methylpropyl)hexanoyl]-N-epsilon-[benzyloxycarbonyl]lysineMethyl Ester

Title product of the preceding Example 0.131 g., 0.0018 mol) was stirredin 5 ml. 4:1 acetic acid:H₂ O for 16 hours, then stripped with toluenechase to yield title product as a white solid, 95 mg.; ¹ H-nmr (CDCl₃)includes delta 3.8 (s, 3H, OCH₃).

EXAMPLE 75N-alpha-[2R,4S,5S-6-Cyclohexyl-5-amino-4-hydroxy-2-(2-methylpropyl)hexanoyl]-N-epsilon-[benzyloxycarbonyl]lysineMethyl Ester

Title product of the preceding Example (95 mg.) was dissolved in 2 ml.of trifluoroacetic acid at 0° C., held for 1 hour, stripped with toluenechase, the residue distributed between 10 ml. of ethyl acetate and 10ml. saturated bicarbonate, the aqueous layer extracted 2×10 ml. freshethyl acetate, and the organic layers combined and washed with brine,dried over MgSO₄ and stripped to yield title product as an oil, 80 mg.;¹ H-nmr (CDCl₃) includes delta 3.8 (s, 3H, OCH₃).

EXAMPLE 76N-alpha-[[2R,4S,5S-6-Cyclohexyl-5-[N-alpha-(2-benzyl-3-phenylpropionyl)-N(imidazole)-(t-butoxycarbonyl)histidyl]amino-4-hydroxy-2-(2-methylpropyl)hexanoyl]]-N-epsilon-[[benzyloxycarbonyl]]lysineMethyl Ester

By the method of Examples 16 and 69, except to use 99:1 CHCl₃ :CH₃ OH aseluant on chromatography, the product of the preceding Example, (80 mg.,0.142 mmol) and the product of Preparation 9 (70 mg., 0.142 mmol) werecoupled to form the present title product, 113 mg. crude, 80 mg.chromatographed; ¹ H-nmr (CDCl₃) include delta 1.6 (s, 9H, C(CH₃)₃).

EXAMPLE 77N-alpha-[[2R,4S,5S-6-Cylcohexyl-5-[N-alpha-(2-benzyl-3-phenylpropionyl)histidyl]amino-4-hydroxy-2-(2-methylpropyl)hexanoyl]]-N-epsilon-[[benzyloxycarbonyl]]lysineMethyl Ester

The product of the preceding Example (80 mg.) was dissolved in 3 ml. of4:1 acetic acid:H₂ O, held for 24 hours, stripped with toluene chase,triturated with 3 ml. 1:1 ether:hexane and filtered to yield titleproduct as a white powder, 40 mg.; ¹ H-nmr (CDCl₃) shows no C(CH₃)₃peaks and includes delta 3.8 (s, 3H, OCH₃).

EXAMPLE 78N-alpha-[[2R,4S,5S-6-Cyclohexyl-5-[N-alpha-(2-benzyl-3-phenylpropionyl)histidyl]amino-4-hydroxy-2-(2-methylpropyl)hexanoyl]]lysineMethyl Ester

The product of the preceding Example (40 mg.) in 1 ml. methanol and 1ml. acetic acid was hydrogenated over 40 mg. of 10% Pd/C for 1.5 hours.The catalyst was recovered by filtration and the filtrate stripped to anoil, chased twice with toluene to yield a white powder which wastriturated with ether and filtered to yield present title product, 18mg.; ¹ H-nmr (CD₃ OD) includes delta 3.8 ppm (s, 3H).

By the method of Example 30, this product is converted to thecorresponding lysine amide derivative.

EXAMPLE 79 S-4-Methyl-2-(t-butoxycarbonylamino)pentanal

By the method of Example 1, N-(t-butoxycarbonyl)leucine methyl ester(28.0 g., 0.114 mol) was converted to present title product, 21.7 g.(88%), as a pale yellow oil; tlc Rf 0.36 (2:3 ethyl acetate:hexane); ¹H-nmr (CDCl₃) delta (90 MHz) 0.97 (d, J=6, 6H), 1.1-1.8 (m, 3H), 1.4 (s,9H), 3.3-5.0 (m, 2H), 9.53 (s, 1H).

EXAMPLE 80 Ethyl4RS,5S-7-Methyl-5-(t-butoxycarbonylamino)-4-hydroxy-2-octynoate

By the method of Example 2, except to use gradient elution with 3:17 to1:4 ethyl acetate:hexane on chromatography, the product of the precedingExample (0.4 g., 0.048 mol) was converted to present title product, 5.45g., tlc Rf 0.40 (3:7 ethyl acetate:hexane); ir (CHCl₃) 3438, 3340, 2233,1711 cm⁻¹ ; ¹ H-nmr J=6, 3H), 1.48 s, 9H), 1.48 (m, 2H), 1.70 (m, 1H),3.3-3.4 (m, 1H), 3.81-3.96 (m, 1H), 4.28 (q, J=7, 2H), 4.45-4.58 (m,1H), 4.68-4.78 (m, 1H).

Anal. Calcd. for C₁₆ H₂₇ NO₅ : C, 61.32; H, 8.68; N, 4.47; Found: C,61.38; H, 8.58; N, 4.42

EXAMPLE 81 4S,5S-7-Methyl-5-(t-butoxycarbonylamino)-gamma-octanolactone

By the methods of Example 3, except to use gradient eluting with 2:3 to1:0 ethyl acetate:hexane on chromatography, the product of the precedingExample was converted to the desired less polar (4S,5S)lactone product,crystallized by trituration with hexane, 3.10 g. (78%); m.p. 76°-77°; ir(CHCl₃) 3439, 1775, 1711 cm⁻¹ ; ¹ H-nmr (CDCl₃) delta (300 MHz) 0.92 (d,J=6, 6H), 1.44 (s, 9H), 1.28-1.81 (m, 3H), 2.06-2.32 (m, 2H), 2.48-2.58(m, 2H), 3.79-3.92 (broad s, 1H), 4.42-4.58 (m, 2H).

Anal. Calcd. for C₁₄ H₂₅ NO₄ : C, 61 97; H, 9.29; N, 5.16; Found: C,62.15; H, 9.26; N, 5.12

The more polar (4R,5S)lactone was also isolated in the chromatography inmuch lower yield and crystallized by hexane trituration, 0.68 g., m.p.113.5°-116° C.

EXAMPLE 822R,4S,5S-7-Methyl-5-(t-butoxycarbonylamino)-2-(2-methyl-2-propenyl)-gamma-octanolactoneMethod A

By the method of Example 4, the title product of the preceding Example(the less polar 4S,5S-epimer; 0.51 g., 0.0019 mol) was converted topresent title product, in major portion, together with its more polar,2S,4S,5S-epimer. The crude products (0.60 g.) were separated bychromatography on a 4.5×20 cm. column of silica gel, eluting with 1 1.each of 1:9, 3:17, 1:5, 1:4, 3:7 and 1:1 ether:hexane, collecting 23 ml.fractions. Fractions 51-85 gave purified title product, 0.21 g.; m.p.128°-132°;

    [alpha].sub.D.sup.30 -23.7° (c=0.529, CH.sub.3 OH)

More polar 2S,4S,5S-epimer was isolated from fractions 89-120, 105 mg.;¹ H-nmr (CDCl₃) includes delta 4.8 (2s, 2H,vinyl protons) and 1.75 (s,3H, vinyl methyl); a portion of this epimer was crystallized by slowevaporation from CH₂ Cl₂ :hexane, providing needle crystals, m.p.99°-101°.

Method B

To a suspension of lithium hexamethyldisilazide at -78° C., prepared bythe dropwise addition of 5.1 ml. (8.11 mmol) a 1.6M solution ofn-butyllithium in hexane to 1.79 ml. (1.39 g., 8.49 mmol) ofhexamethyldisilazane in 3.5 ml. of THF at 0° C., was added dropwise asolution of 1.00 g. (3.69 mmol) of 4S,5S-lactone of the precedingExample in 3 ml. of THF. At the end of the addition the mixture becameclear and it was allowed to stir an additional 15 minutes at -78° C. Asolution of i0 0.548 g. (4.06 mmol) of freshly distilled methallylbromide in 2 ml. of THF was then added dropwise over 5 minutes, and themixture was allowed to slowly warm to -40° C. over 2 hours before beingquenched with 2 ml. of saturated NH₄ Cl. After warming to roomtemperature the reaction mixture was partitioned between 30 ml. of etherand 30 ml. of 10% citric acid The organic layer was separated and washedwith 10% citric acid (3×30 ml.) and saturated NaHCO₃, dried (MgSO₄), andevaporated to 1.11 g. of a crude mixture of cis(2S) and trans(2R). Theselactones were separated on 88 g. of silica gel with an ether-hexane (1:9to 3:7) eluant. The fractions containing the less polar trans(2S)lactone [tlc Rf 0.55 (1:1 ether:hexane)] were combined andevaporated to 0.613 g. (51%) of a white solid, m.p. 132°-135° C. Minorimpurities (as indicated by tlc) were removed by trituration in hexaneto afford 0.562 g. (47%) of analytically pure title 2R,4S,5S-lactone,m.p. 133°-135° C. Crystals suitable for X-ray analysis were prepared byslow evaporation from hexane-methylene chloride. ¹ H-nmr (CDCl₃) delta(250 MHz) 0.90 (J, J=6, 3H), 0.92 (d, J=6, 3H), 1.42 (s, 9H), 1.70 (s,3H), 1.92-2.15 (m, 2H), 2.26-2.39 (m, 1H), 2.57 (dd, J=15 and 3, 1H),2.72-2.88 (m, 1H), 3.77-3.90 (m, 1H), 4.34 (d, J=9, 1H), 4.33-4.51 (m,1H), 4.70 (s, 1H), 4.81 (s, 1H): ¹³ C-nmr (75 mHz) delta 21.8, 23.0,24.7, 28.3, 30.0, 37.9, 39.5, 41.8, 51.7, 79.8, 80.7, 112.8, 141.9,156.0, 179.3; IR (CHCl₃) 3439, 1768, 1712, 1654 cm⁻¹ ; [alpha]_(D)-25.0° (C=0.5, CH₃ OH). Anal. Calcd. for C₁₈ H₃₁ NO₄ : C, 66.43; H,9.60; N, 4.30. Found: C, 66.47; H, 9.59; N, 4.27. Single crystal X-rayanalysis proved that the structure and stereochemical assignment of thiscompounds was correct.

The fractions containing the more polar cis(2S) lactone (tlc Rf 0.441:11 ether:hexane) were combined and evaporated to 39 mg (3%) of a whitesolid, mp 96°-98° C.; ¹ H-nmr (CDCl₃) delta (250 MHz) 0.92 (d, J=6, 6H),1.43 (s, 9H), 1.72 (s, 3H), 2.02-2.14 (m, 1H), 2.23-2.36 (m, 1H),2.60-2.87 (m, 2H), 3.74-3.89 (m, 1H), 4.35-4.47 (m, 2H), 4.69 (s, 1H),4.78 (s, 1H); ¹³ nmr (75 MHz) delta 21.9, 22.0, 23.0, 24.8, 28.3, 30.7,38.8, 38.9, 42.3, 50.1, 79.6, 80.4, 112.6, 142.0, 155.9, 178.7; IR(CHCl₃) 3443, 1774, 1714, 1656 cm⁻¹ ; [alpha]_(D) -0.6° [C=0.5, CH₃ OH).Anal. Calcd. for C₁₈ H₃₁ NO₄ : C, 66.43; H, 9.60; N, 4.30. Found: C,66.94; H, 9.45; N, 4.27.

EXAMPLE 832R,4S,5S-7-Methyl-5-(t-butoxycarbonylamino)-2-(2-methylpropyl)-gamma-octanolactone

An ethyl acetate (10 ml) solution of 438 mg. (1.35 mmol) of the titlelactone of the preceding Example containing 44 mg. of 10% Pd/C washydrogenated on a Parr Shaker apparatus at 50 psi for 2 hours. Afterfiltration of the catalyst and evaporation of the solvent, 437 mg. (99%)of present title product was obtained as a white solid, mp 130°-131° C.¹ H-nmr (CDCl₃) delta (300 MHz) 0.84-0.97 (m, 12H), 1.41 (s, 9H),1.86-1.96 (m, 1H), 2.30-2.42 (m, 1H), 2.56-2.68 (m, 1H), 3.76-3.89 (m,1H), 4.35 [d, J=8, 1H), 4.45 (broad t, 1H); ¹³ C-nmr (75 Hz) delta 21.3,21.8, 22.9, 23.0, 24.8, 26.1, 28.3, 31.0, 37.7, 40.5, 41.9, 51.7, 79.8,80.5, 156.0, 180.3; IR (CHCl₃) 3439, 1769, 1713 cm⁻¹ ; [alpha]_(D)-32.1° (C=1.0, CH₃ OH). Anal. Calcd. for C₁₈ H₃₃ NO₄ : C, 66.02; H,10.16; N, 4.28. Found: C, 66.07; H, 10.03; N, 4.05.

By the various procedures of the preceding Examples, the present productis converted to analogous renin inhibiting products wherein R¹ is2-methylpropyl rather than cyclohexylmethyl.

EXAMPLE 84 Benzyl3-[2R,4S,5S-6-Cyclohexyl-5-[t-butoxycarbonylamino)-4-(2-tetrahydropyranyloxy)-2-(2-methylpropyl)hexanoylamino]propionate

The product of Example 8 (0.30 g., 0.64 mmol) and benzyl beta-alaninehydrochloride (0.15 g., 0.70 mmol) were combined in 5 ml. CH₂ Cl₂ at 0°C. N-Methylmorpholine (0.154 ml., 1.41 mmol) was added with stirring,followed by diethyl cyanophosphonate (0.109 ml., 0.70 mmol). Afterstirring 16 hours at 0° and 1 hour at room temperature, the mixture wasstripped to dryness, and the residue taken up in ethyl acetate 20 ml.),extracted 2×20 ml. saturated NaHCO₃, 1×20 ml. H₂ O and 1×20 ml. brine,dried (MgSO₄) and stripped to 0.394 g. of crude product, chromatographedon 45 g. of silica gel with 99:1 CHCl₃ :CH₃ OH as eluant to producepurified present title product, 0.30 g.; tlc Rf 0.8 (19:1:CHCl₃ :CH₃OH).

EXAMPLE 85 Benzyl 3-[2R,4S,5S-6-Cyclohexyl-5-(t-butoxycarbonylamino)-4-hydroxy-2-(2-methylpropyl)-hexanoylamino]propionate

The product of the preceding Example (300 mg.) was stirred with 10 ml.4:1 acetic acid:H₂ O for 16 hours, stripped under high vacuum and chasedwith toluene to yield title product, 273 mg.

EXAMPLE 86 Benzyl3-[2R,4S,5S-6-Cyclohexyl-5-amino-4-hydroxy-2-(2-methylpropyl)hexanoylamino]propionateHydrochloride

The product of the preceding Example (273 mg.) was dissolved in 3 ml. ofether and treated with 3 ml. 3.7N HCl in dioxane at 0° C. After 4 hoursat 0°, the reaction was stripped and triturated with ether-hexane toyield title product as white solids, 248 mg.

EXAMPLE 87 Benzyl3-[2R,4S,5S-6-Cyclohexyl-5-[N-alpha-(N-(t-butoxycarbonyl)phenylalanyl)-N(imidazole)-t-butoxycarbonyl)histidyl]amino-4-hydroxy-2-(2-methylpropyl)hexanoylamino]propionate

Except to use 39:1 CHCl₃ :CH₃ OH as eluant on chromatography, the methodof Example 84 was used to couple the product of the preceding Example(248 mg, 0.589 mmol) and the product of Preparation 1 (357 mg., 0.707mmol) in the presence of N-methylmorpholine (0.155 ml., 1.413 mmol) anddiethyl cyanophosphonate 0.11 ml., 0.707 mmol), thereby producingpurified title product, 0.225 g.; tlc 0.5 (9:1 CHCl₃ :CH₃ OH).

EXAMPLE 88 Benzyl3-[2R,4S,5S-6-Cyclohexyl-5-[N-(t-butoxycarbonyl)phenylalanylhistidyl]amino-4-hydroxy-2-(2-methylpropyl)hexanoylamino]propionate

By the method of Example 85, the product of the preceding Example (342mg.) was converted to present title product, 305 mg.; tlc Rf 0.2 (9:1CHCl₃ :CH₃ OH).

EXAMPLE 893-[2R,4S,5S-6-Cyclohexyl-5-[N-(t-butoxycarbonyl)-phenylalanylhistidyl]amino-4-hydroxy-2-(2-methylpropyl)hexanoylaminopropionicAcid

Using 10 ml. of pure methanol as solvent, the product of the precedingExample (0.305 g.) was hydrogenated according to Example 13. Followingcatalyst recovery the filtrate was stripped to yield present titleproduct, 0.234 g.; ¹ H-nmr (CDCl₃) includes delta 1.5 ppm (s, 9H).

EXAMPLE 902R,4S,5S-6-Cyclohexyl-5-(t-butoxycarbonylamino)-4-hydroxy-2-(2-methyl-2-propenyl)-N-methylhexanamide

The trans(2R) title product of Example 4 (0.20 g., 0.55 mmol) wasdissolved in 10 ml. CH₃ OH, cooled to 0° C., and the solution saturatedwith CH₃ NH₂. After holding at 0° for 60 hours, the mixture was strippedand the solid residue triturated with hexane to yield title product,0.220 g.

EXAMPLE 912R,4S,5S-6-Cyclohexyl-5-amino-4-hydroxy-2-(2-methyl-2-propenyl)-N-methylhexanamide

Product of the preceding Example (0.220 g.) was stirred with 2 ml. oftrifluoroacetic acid at -11° C. for 0.5 hour. The reaction mixture wasstripped and the residue taken up in 5 ml. ethyl acetate, washed with 5ml. saturated NaHCO₃ and 5 ml. of brine, dried (MgSO₄) and stripped toyield title product, 0.169 g.

EXAMPLE 922R,4S,5S-6-Cyclohexyl-5-[N-alpha-(N-(t-butoxycarbonylphenylalanyl)-N(imidazole)-(t-butoxycarbonyl)-histidyl]amino-4-hydroxy-2-2-methyl-2-propenyl)-propenylN-methylhexanamide

The product of the preceding Example (169 mg., 0.57 mmol) and theproduct of Preparation 1 (288 mg., 0.57 mmol) were combined in 15 ml.CH₂ Cl₂ with 1-hydroxybenzotriazole (77 mg., 0.57 mmol) anddicyclohexylcarbodiimide (118 mg., 0.57 mmol) at 0°. After stirring atambient temperature for 18 hours, dicyclohexyl urea was recovered byfiltration, the filtrate stripped, and the resulting residue taken up in10 ml. ethyl acetate, washed 1×5 ml. saturated NaHCO₃, 1×5 ml. 5% HCland 1×5 ml brine, dried (MgSO₄), stripped (0.329 g.) and chromatographedon 35 g. silica gel with 40:1 CHCl₃ :CH₃ OH as eluant to yield purifiedtitle product, 0.129 g.

EXAMPLE 932R,4S,5S-6-Cyclohexyl-5-[N-(t-butoxycarbonyl)phenylalanylhistidyl]amino-4-hydroxy-2-(2-methyl-2-propenyl)-N-methylhexanamide

By the method of Example 85, with final trituration of the solid productwith ether, the product of the preceding Example (0.129 g.) wasconverted to present title product, 75 mg.; ¹ H-nmr (CDCl₃) includesdelta 1.5 ppm (s, 9H).

EXAMPLE 94 Ethyl4-[2R,4S,5S-Cyclohexyl-5-(t-butoxycarbonylamino)-4-(2-tetrahydropyranyloxy)-2-(2-methylpropylhexanoylamino]butyrate

Without chromatography, the method of Example 84 was employed to couplethe product of Example 8 (300 mg., 0.64 mmol) with ethyl 4-aminobutyratehydrochloride (118 mg., 0.70 mmol) to yield title product, 385 mg.

EXAMPLE 95 Ethyl4-2R,4S,5S-6-Cyclohexyl-5-(t-butoxycarbonylamino)-4-hydroxy-2-(2-methylpropyl)hexanoylamino]butyrate

The product of the preceding Example (0.385 g.) with treated 4:1 aceticacid:H₂ O according to Example 85, crude, lactone containing product(330 mg.) was isolated by freeze drying and title product isolated bychromatography on silica gel, eluting lactone with 1:1 ether:hexane andtitle product with 19:1 CHCl₃.CH₃ OH, 161 mg.; tlc Rf 0.6 (9:1 CHCl₃:CH₃ OH).

EXAMPLE 96 Ethyl4-[2R,4S,5S-6-Cyclohexyl-5-amino-4-hydroxy-2-(2-methylpropyl)hexanoylamino]butyrateHydrochloride

The product of the preceding Example (0.161 g.) was dissolved in 5 ml.ether and cooled to 0° C. 7.4N HCl in dioxane (3 ml.) was added and themixture stirred at 0° C. for 3.5 hours, then stripped and dried underhigh vacuum to yield title product, 0.141 g., tlc Rf 0.1 (9:1 CHCl₃ :CH₃OH).

EXAMPLE 97 Ethyl4-[2R,4S,5S-6-Cyclohexyl-5-[N-alpha-(N-(t-butoxycarbonyl)phenylalanyl)-N(imidazole)-(t-butoxycarbonyl)histidyl]amino-4-hydroxy-2-(2-methylpropyl)hexanoylamino]butyrate

By the method of Example 87, the product of the preceding Example (140mg., 0.323 mmol) and the product of Preparation 1 (179 mg., 0.355 mmol)were coupled, isolated and purified to yield title product, 136 mg. Bythe method of Example 88, this product was converted to ethyl4-[2R,4S,5S-6-cyclohexyl-5-[N-(t-butoxycarbonyl)phenylalanyl-histidyl]amino-4-hydroxy-2-(2methylpropyl)hexanoylamino]butyrate.

EXAMPLE 982R,4S,5S-6-Cyclohexyl-5-(t-butoxycarbonylamino)-2-(p-chlorobenzyl)-gamma-hexanolactone

By the method of Example 39, the less polar 4S,5S-product of Example 3(2.5 g., 0.008 mol) and 4-chlorobenzyl bromide (1.81 g., 0.0088 mol)were converted to present chromatographed title product as a colorlessgum, 1.91 g.; tlc Rf 0.6 (3:1 hexane:ethyl acetate) 0.7 (2:1hexane:ethyl acetate with 1% acetic acid).

EXAMPLE 992R,4S,5S-6-Cyclohexyl-5-(t-butoxycarbonylamino)-2-(p-methylbenzyl)-gamma-hexanolactone

Using 5:1 hexane:ethyl acetate as eluant on chromatography, the methodof Example 39 was used to convert the 4S,5S product of Example 3 (1.5g., 0.0048 mol) and alpha-bromo-p-xylene (0.98 g., 0.0053 mol) topresent title product as a white gum, 0.985 g.; tlc Rf 0.55 (2:1hexane:ethyl acetate), 0.75 (1:1 hexane:ethyl acetate).

EXAMPLE 1002R,4S,5S-6-Cyclohexyl-5-(t-butoxycarbonylamino)-2-(p-methoxybenzyl)-gamma-hexanolactone

By the method of Example 99, the 4S,5S-product of Example 3 (3.79 g.,0.0118 mol) and p-methoxybenzylbromide (2.61 g., 0.130 mol) wereconverted to chromatographed title product, as a white gum, 1.06 g.; tlcRf 0.4 (3:1 hexane:ethyl acetate).

EXAMPLE 1012R,4S,5S-6-Cyclohexyl-5-t-butoxycarbonylamino)-2-(3,4-dichlorobenzyl)-gamma-hexanolactone

By the method of Example 39, using 3:1 hexane:ethyl acetate as eluant onchromatography, the 4S,5S-product of Example 3 (2.0 g., 0.0064 mol) and3,5-dichlorobenzyl bromide (1.68 g., 0.007 mol) were converted to titleproduct as a clear gum, 1.36 g.; tlc Rf 0.22 (3:1 hexane:ethyl acetate),0.9 (1:2 hexane:ethyl acetate with 1% acetic acid).

EXAMPLE 1022R,4S,5S-6-Cyclohexyl-5-(t-butoxycarbonylamino)-2-(o-chlorobenzyl)-gamma-hexanolactone

By the method of Example 39, using 6:1 hexane:ethyl acetate as eluant onchromatography, the 4S,5S-product of Example 3 (2.0 g., 0.0064 mol) ando-chlorobenzyl chloride (1.44 g., 0.007 mol) were converted to titleproduct as a clear gum, 1.51 g.; tlc Rf 0.75 (3:1 hexane:ethyl acetate),0.65 (2:1 hexane:ethyl acetate with 1% acetic acid).

EXAMPLE 1032R,4S,5S-6-Cyclohexyl-5-(t-butoxycarbonylamino)-2-(m-chlorobenzyl)-gamma-hexanolactone

By the method of the preceding Example, 4S,5S-product of Example 3 (2.0g., 0.0064 mol) and m-chlorobenzyl chloride (0.92 ml., 1.44 g., 0.007mol) were converted to chromatographed title product as a colorless gum,2.11 g.; tlc Rf 0.4 (3:1 hexane:ethyl acetate), 0.75 (2:1 hexane:ethylacetate with 1% acetic acid).

EXAMPLE 1042R,4S,5S-6-Cyclohexyl-5-(t-butoxycarbonylamino)-4-hydroxy-2-(p-chlorobenzyl)-N-methylhexanamide

Using a reaction time of 2 hours at ambient temperature, without hexanetrituration, the procedure of Example 90 was used to convert the productof Example 98 (1.0 g.) to present title product as white solids, 1.07g.; tlc Rf 0.75 (2:1 hexane:ethyl acetate with 1% acetic acid), 0.6(18:2:1 CH₂ Cl₂ :C₂ H₅ OH:acetic acid).

EXAMPLE 1052R,4S,5S-6-Cyclohexyl-5-(t-butoxycarbonyl)-4-hydroxy-2-(p-methylbenzyl)-N-methylhexanamide

Using the method of the preceding Example, chromatographing the producton silica gel with 1:1 hexane:ethyl acetate as eluant, the product ofExample 99 (0.885 g.) was converted to present title product, 0.518 g.;tlc Rf 0.1 1:1 ethyl acetate:hexane), 0.55 (18:2:1 CHCl₃ :C₂ H₅OH:acetic acid).

EXAMPLE 1062R,4S,5S-6-Cyclohexyl-5-(t-butoxycarbonylamino)-4-hydroxy-2-(p-methoxybenzyl)-N-methylhexanamide

By the method of Example 4, the product of Example 100 (1.06 g.) wasconverted to present title product as off-white solids, 1.07 g.; tlc Rf0.2 (2:1 ethyl acetate:hexane with 1% acetic acid), 0.8 (9:1 CH₂ Cl₂ :C₂H₅ OH with 1% concentrated NH₄ OH).

EXAMPLE 1072R,4S,5S-5-Cyclohexyl-4-(t-butoxycarbonylamino)-4-hydroxy-2-(3,4-dichlorobenzyl)-N-methylhexanamide

By the method of Example 104, the product of Example 101 (1.36 g.) wasconverted to present product as a white gum, 1.39 g.; tlc Rf 0.25 (1:1hexane:ethyl acetate with 1% acetic acid), 0.55 (9:1 CH₂ Cl₂ :C₂ H₅ OHwith 1% concentrated NH₄ OH.

EXAMPLE 1082R,4S,5S-6-Cyclohexyl-5-(t-butoxycarbonylamino)-4-hydroxy-2-(o-chlorobenzyl)-N-methylhexanamide

Except to use a reaction time of 16 hours at ambient temperature, themethod of Example 104 was used to convert the product of Example 102(1.51 g.) to present product as a white gum, 1.56 g.; tlc Rf 0.09 (2:1hexane:acetic acid with 1% acetic acid).

EXAMPLE 1092R,4S,5S-6-Cyclohexyl-5-(t-butoxycarbonylamino)-4-hydroxy-2-(m-chlorobenzyl)-N-methylhexanamide

By the method of Example 104, the product of Example 103 (2.11 g.) wasconverted to present title product as a white gum, 1.68 g.; tlc Rf 0.25(2:1 hexane:ethyl acetate with 1% acetic acid), 0.5 (9:1 CH₂ Cl₂ :C₂ H₅OH with 1% acetic acid).

EXAMPLE 1102R,4S,5S-6-Cyclohexyl-5-amino-4-hydroxy-2-(p-chlorobenzyl)-N-methylhexanamideHydrochloride

The product of Example 104 (1.07 g.) was stirred under N₂ in 10 ml. of3.78N HCl in dioxane for 10 minutes, stripped. The residue was 3 timescombined with 10 ml. of ether and restripped, producing title product asa solid, 1.11 g., tlc Rf 0.18 (18:2:1 CH₂ Cl₂ :C₂ H₅ OH:acetic acid).

EXAMPLE 1112R,4S,5S-6-Cyclohexyl-5-amino-4-hydroxy-2-(p-methylbenzyl)-N-methylhexanamideHydrochloride

By the method of the preceding Example, the product of Example 105(0.454 g.) was converted to present title product, 0.4 g.; tlc Rf 0.1(18:2:1 CHCl₃ :C₂ H₅ OH acid).

EXAMPLE 1122R,4S,5S-6-Cyclohexyl-5-amino-4-hydroxy-2-(p-methoxybenzyl)-N-methylhexanamideHydrochloride

By the method of Example 110, the product of Example 106 (1.07 g.) wasconverted to present title product in quantitative yield (weight yield1.14 g., 0.22 g., greater than theory); tlc Rf 0.2 (9:1 CH₂ Cl₂ :C₂ H₅OH with 1% concentrated HN₄ OH).

EXAMPLE 1132R,4S,5S-6-Cyclohexyl-5-amino-4-hydroxy-2-[3,4-dichlorobenzyl)-N-methylhexanamideHydrochloride

By the method of Example 110, the product of Example 107 (1.38 g.) wasconverted to present title product in quantitative yield (weight 1.52g., 0.30 g. greater than theory); tlc Rf 0.25 (CH₂ Cl₂ :C₂ H₅ OH 9:1with 1% concentrated NH₄ OH).

EXAMPLE 1142R,4S,5S-6-Cyclohexyl-5-amino-4-hydroxy-2-(o-chlorobenzyl)-N-methylhexanamideHydrochloride

By the method of Example 110, the product of Example 108 (1.56 g.) wasconverted to present title product, 1.4 g.; tlc Rf 0.1 (9:1 CH₂ Cl₂ :C₂H₅ OH with 1% acetic acid).

EXAMPLE 1152R,4S-,5S-6-Cyclohexyl-5-amino-4-hydroxy-2-(m-chlorobenzyl)-N-methylhexanamideHydrochloride

By the method of Example 110, the product of Example 109 (1.67 g.) wasconverted to present title product in quantitative yield (0.23 g.greater than theory); tlc 0.1 (9:1 CH₂ Cl₂ :C₂ H₅ with 1% acetic acid).

EXAMPLE 1162R,4S,5S-6-Cyclohexyl-5-[N-(t-butoxycarbonyl)-phenylalanyl-N(imidazole)-(t-butoxycarbonyl)-histidyl]amino-4-hydroxy-2-(p-chlorobenzyl)-N-methylhexanamide

Under nitrogen the product of Example 110 (1.11 g., 0.0023 mol) wasstirred with 8 ml. CH₂ Cl₂. Added in sequence were triethylamine (0.42ml., 0.0030 mol), the product of Preparation 1 (1.20 g., 0.0024 mol),1-hydroxybenzotriazole (0.583 g., 0.0038 mol) anddicyclohexylcarbodiimide (0.495 g., 0.0024 mol) and the mixture stirredfor 24 hours. The reaction mixture was diluted with 25 ml. CH₂ Cl₂,washed with 2×12 ml. 1N NaOH and 1×12 ml. saturated NaCl, dried (Na₂SO₄) and stripped to a foam, 1.31 g. The latter was chromatographed onsilica gel with 19:1 CH₂ Cl₂ :C₂ H₅ OH as eluant to yield purified titleproduct, 0.70 g.; tlc Rf 0.65 (18:2:1 CH₂ Cl₂ :C₂ H₅ OH:acetic acid).

EXAMPLE 117 2R,4S,5S-6-Cyclohexyl-5-[N-(t-butoxycarbonyl)-phenylalanyl-N(imidazole)-(t-butoxycarbonyl)-histidyl]amino-4-hydroxy-2-(p-methylbenzyl)-N-methylhexanamide

Except to combine the reagents at 0° C., then allowing the reactionmixture to warm, and to use 5:1 CH₂ C₂ :C₂ H₅ OH as eluant onchromatography, the method of the preceding Example was used to convertthe product of Example 111 (0.4 g.) to present title product, 0.60 g.;tlc Rf 0.9 (18:2:1 CH₂ Cl₂ :C₂ H₅ OH:acetic acid).

EXAMPLE 1182R,4S,5S-6-Cyclohexyl-5-[N-(t-butoxycarbonyl)-phenylalanyl-N(imidazole)-(t-butoxycarbonyl)-histidyl]amino-4-hydroxy-2-(p-methoxybenzyl)-N-methylhexanamide

By the method of the preceding Example, using 20:1 CH₂ Cl₂ :C₂ H₅ OH aseluant on chromatography, the product of Example 112 (0.92 g., correctedfor purity) was converted to present title product as a white solid,0.73 g.; tlc Rf 0.7 (9:1 CH₂ Cl₂ :C₂ H₅ OH), 0.85 (9:1 CH₂ Cl₂ :CH₃ OHwith 1% concentrated NH₄ OH).

EXAMPLE 1192R,4S,5S-6-Cyclohexyl-5-[N-(t-butoxycarbonyl)-phenylalanyl-N(imidazole)-t-butoxycarbonyl)histidyl]amino-4-hydroxy-2-(3,4-dichlorobenzyl)-N-methylhexanamide

By the method of Example 117, using 24:1 CH₂ Cl₂ :C₂ H₅ OH as eluant onchromatography, the product of Example 113 (1.22 g., corrected forpurity) was converted to present product, 1.08 g.; tlc Rf 0.75 (9:1CHCl₃ :C₂ H₅ OH).

EXAMPLE 1202R,4S,5S-6-Cyclohexyl-5-[N-(t-butoxycarbonyl)-phenylalanyl-N(imidazole)-(t-butoxycarbonyl)histidyl]-amino-4-hydroxy-2-(o-chlorobenzyl)-N-methylhexanamide

By the method of Example 119, the product of Example 114 (1.33 g.,corrected for purity) was converted to chromatographed title product asa white solid, 1.02 g.; tlc Rf 0.55 (9:1 CH₂ Cl₂ :ethyl acetate with 1%acetic acid).

EXAMPLE 1212R,4S,5S-6-Cyclohexyl-5-[N-(t-butoxycarbonyl)-phenylalanyl-N(imidazole)-(t-butoxycarbonyl)-histidyl]amino-4-hydroxy-2-(m-chlorobenzyl)-N-methylhexanamide

By the method of Example 119, the product of Example 115 (1.45 g.,corrected for purity) was converted to chromatographed title product asa white gum, 1.68 g.; tlc Rf 0.62 (9:1 CH₂ Cl₂ :C₂ H₅ OH with 1% aceticacid).

EXAMPLE 1222R,4S,5S-6-Cyclohexyl-5-(t-butoxycarbonylphenylalanyl-histidyl)amino-4-hydroxy-2-(m-chlorobenzyl)-N-methylhexanamide

The product of Example 116 (0.25 g.) was combined with 2 ml. glacialacetic acid and 0.5 ml. H₂ O and stirred under N₂ for 8 hours, thenstripped to dryness, and the residue triturated 3×0.5 ml. toluene, thenslurried 3×5 ml. ether, restripping each time, to yield 83 mg. of crudeproduct. The latter was purified on 5 g. of bondedphase--octadecylsilane (C₁₈) reverse phase packing in a 10 mm i.d. flashcolumn with 1:1 CH₃ OH:H₂ O as the initial mobile phase. The crude wasto the column. The column was eluted with 4 column volumes 1:1 CH₃ OH:H₂O, 2 column volumes 6:4 CH₃ OH:H₂ O, 2 column volumes 7:3 CH₃ OH:H₂ O, 2column volumes 8:2 CH₃ OH:H₂ O to remove more polar impurities. Titleproduct came off in 2 column volumes of 9:1 CH.sub. 3 OH:H₂ O, recoveredas white solids, 30 mg.; DMSO-d₆) includes delta (ppm) 1.28 (s, 9H,t-butyl) and 6.99 (d, J=7 Hz, 1H, imidazole hydrogen).

EXAMPLE 1232R,4S,5S-6-Cyclohexyl-5-(t-butoxycarbonylphenylalanyl-histidyl)amino-4-hydroxy-2-(p-methylbenzyl)-N-methylhexanamide

By the method of the preceding Example, except that product eluted alittle later, in 2 column volumes of 100% CH₃ OH, the product of Example117 (100 mg.) was converted to purified title product as white solids,41 ¹ H-nmr (300 MHz, DMSO-d₆) delta (ppm) includes 1.25 (s, 9H,t-butyl), 6.83 (d, J=12 Hz, 1H, imidazole hydrogen).

EXAMPLE 1242R,4S,5S-6-Cyclohexyl-5-[t-butoxycarbonylphenylalanyl-histidyl)amino-4-hydroxy-2-(p-methoxybenzyl)-N-methylhexanamide

By the method of the preceding Example, the product of Example 118(0.362 g.) was converted to present purified title product as whitesolids, 0.126 g.; tlc Rf 0.55 (9:1 CH₂ Cl₂ :CH₃ OH with 1% concentrated¹ H-nmr (300 MHz, DMSO-d₆) delta (ppm) includes 1.27 (s, 9H, t-butyl),2.42 (d, J=4 Hz, 3H, NCHl₃) and 3.66 (s, 3H, OCH₃).

EXAMPLE 1252R,4S,5S-6-Cyclohexyl-5-(t-butoxycarbonyl-phenylalanyl-histidyl)amino-4-hydroxy-2-(3,4-dichlorobenzyl)-N-methylhexanamide

By the method of Example 122, without chromatography, the product ofExample 119 (1.08 g.) was converted to present title product as whitesolids, 0.90 g.; ¹ H-nmr (300 MHz, DMSO-d₆) delta (ppm) includes 1.29(s, 9H, t-butyl), 2.47 (d, J=5Hz, 3H, NCH₃).

EXAMPLE 1262R,4S,5S-6-Cyclohexyl-5-(t-butoxycarbonylphenylalanyl-histidyl)amino-4-hydroxy-2-(o-chlorobenzyl)-N-methylhexanamide

By the procedure of the preceding Example, the product of Example 120(1.01 g.) was converted to crude title product (0.48 g.), purified bychromatography on silica gel using 9:1 CH₂ Cl₂ :CH₃ OH as eluant, 0.22g.; tlc Rf 0.5 (9:1 CH₂ Cl₂ :CH₃ OH with 1% concentrated NH₄ OH); ¹H-nmr (300 MHz, DMSO-d₆ ) delta (ppm) includes 1.27 (s, 9H, t-butyl),2.45 (d, J=4 Hz, 3H, NCH₃).

EXAMPLE 1272R,4S,5S-6-Cyclohexyl-5-(t-butoxycarbonylphenylalanyl-histidyl)amino-4-hydroxy-2-(m-chlorobenzyl)-N-methylhexanamide

By the procedure of the preceding Example, the product of Example 121(0.94 g.) was converted to chromatographed title product as whitesolids, 0.58 g.; tlc Rf 0.15 (9:1 CH₂ Cl₂ :C₂ H₅ OH with 1% aceticacid); ¹ H-nmr (300 MHz, DMSO-d₆) delta (ppm) includes 1.27 (s, 9H,t-butyl), 2.45 (d, J=5 Hz, 3H, NCH₃).

PREPARATION 1N-alpha-[N-t-Butoxycarbonylphenylalanyl]N(imidazole)-(t-butoxycarbonyl)histidine

A slurry of 36.4 g. L-histidine methyl ester dihydrochloride indichloromethane (1000 ml.) was cooled to 5° and treated with 52 ml.triethylamine. After 10 minutes 40 g. t-butoxycarbonyl-phenylalanine wasadded followed by 1-hydroxybenzotriazole (30.6 g.) and, after another 5minutes, dicyclohexylcarbodiimide (30.8 g.). The mixture was thenfiltered and washed with dichloromethane. The combined filtrate and washliquor was stripped and the residue was dissolved in 1000 ml. ethylacetate. After 10 minutes of stirring the mixture was filtered and thefiltrate was washed with 1N NaOH (3×150 ml.) and brine, dried overMgSO₄, and concentrated giving 45.9 g. of intermediatet-butoxycarbonyl-phenylalanyl-histidine methyl ester as a colorlesssolid. Without further purification, 40 g. of this intermediate solidwas dissolved in 600 ml. methanol and 200 ml. water was added. Themixture was chilled to 0° and treated with 40 g. anhydrous potassiumcarbonate, stirred at 15°-20° for 2.5 hours then at 28° for 1.5 hours,cooled to 10°, and adjusted to pH 4.2 with 12N HCl. The resultingsolution was concentrated to about 250 ml. and 70 ml. water was added,followed by 660 ml. dioxane. At 0° the pH was brought to 13.5 and 29 ml.di-(t-butyl)dicarbonate was added. After 30 minutes (during which timethe temperature was raised to 20° ) the pH had dropped to 9.5, and 10ml. di-(t-butyl)dicarbonate was added. After another 1 hour the pH was8.0 and the reaction was complete. The mixture was concentrated toremove dioxane, 300 ml. water was added, and the mixture was washedtwice with ether. Ethyl acetate (500 ml.) was added and at 10° the pHwas brought to 1.2 with conc. HCl. The organic layer was separated andthe aqueous layer was washed twice with ethyl acetate. The ethyl acetatelayers were combined, washed with water, brine, dried over Na₂ SO₄, andconcentrated to give after several coevaporations with ether and dryingat 25° C. to constant weight a colorless foam, weight 44 g. HPLC at60/40 MeCN/pH 2.1 0.1M phosphate on Zorbax 25 cm×4.6 mm at 214 nm, 1.5ml/min., retention time 3.23 minutes (94% of the total absorbance to 10minutes); ¹ H-nmr (CDCl₃) ppm: 1.40 (s, 9H, C(CH₃)₃), 1.60 (s, 9H,C(CH₃)hd 3), 3.0-3.4 (m, 4H, CH₂), 4.40 (m, 1H), 4.71 (m, 1H), 5.32 (m,1H), 6.89 (br, 1H), 8.13 (s, 1H), 7.1-7.4 (m, 7H).

PREPARATION 2 1,5-Di(benzyloxycarbonylamino)pentane

Under N₂, 1,5-diaminopentane (2.86 ml., 2.5 g., 0.0245 mol) wasdissolved in 50 ml. CH₂ Cl₂ and cooled to 0°. With stirring,benzyloxycarbonyl chloride (3.67 ml., 4.17 g., 0.0245 mol) was addeddropwise, maintaining the temperature near 0°. The mixture was warmed toroom temperature, washed 2×50 ml. 5% HCl, 1×50 ml. saturated NaHCO₃, and1×50 ml. brine, dried (MgSO₄) and stripped to yield title product as awhite solid., 4 g.; ¹ H-nmr (CDClhd 3) includes delta 5.2 ppm (s, 4H,two benzyl CH₂ groups). The yield is greatly enhanced by use of twomolar equivalents of the acid chloride.

PREPARATION 3 5-(Benzyloxycarbonylamino)pentylamine Hydrobromide

Under nitrogen, the product of the preceding Preparation 4.0 g., 0.0108mol) was slurried in CH₂ Cl₂ (11 ml.) and ether (5.4 ml.). 30% HBr inacetic acid (2.7 ml.) was added with stirring. Solids began toprecipitate from the resulting solution within 2 minutes. After 20minutes, 5 ml. ether was added and title product recovered by filtrationwith ether wash, 2.7 g., ¹ H-nmr (CD₃ OD) includes delta 5.2 ppm (s, 2H,benzyl CH₂).

PREPARATION 4 N-alpha-Methylhistidine Methyl Ester Dihydrochloride

N-alpha-Methylhistidine (Reinhold et al., J. Med. Chem. 11, p. 258,1968, 4.0 g., 0.0195 mol) was slurried in 40 ml. methanol at 0°, spargedwith dry HCl for 8 minutes (solution occurred after 3 minutes), heatedto reflux for 4 hours, and co-stripped with ether to yield title productas a white powder, 4.94 g.

PREPARATION 5N-alpha-Methyl-N-alpha-[N-(t-butoxycarbonyl)-phenylalanyl)histidineMethyl Ester

The product of the preceding Preparation (1.6 g., 0.0087 mol),N-(t-butoxycarbonyl)phenylalanine (2.55 g., 0.096 mol),1-hydroxybenzotriazole (2.44 g., 0.016 mol) and dicyclohexylcarbodiimide(1.98 g., 0.0096 mol) were combined in 10 ml. of CH₂ Cl₂ at 0° for 4hours. The reaction mixture was diluted with 10 ml. fresh CH₂ Cl₂,filtered to yield a first solid, the filtrate stripped, the residuetaken up in 15 ml. ethyl acetate, refiltered to yield a second solid,the second filtrate washed 2×7.5 ml. 1N NaOH, 1×7.5 ml. H₂ O, a thirdsolid removed by filtration after cooling to 0°, and the third filtrateconcentrated to dryness and the residue triturated with ethyl acetate toform a fourth solid. The first and second solids (DCU) were discarded.The third and fourth solids, containing very little DCU, were combinedto yield 2.47 g. of title product.

PREPARATION 6N-alpha-Methyl-N-alpha-[N-t-butoxycarbonyl)phenylalanyl]histidine

The product of the preceding Preparation (2.32 g., 0.054 mol) in 70 ml.acetone and 20 ml. H₂ O was hydrolyzed for 3.5 hours with 1.1N NaOH (5.4ml., 1.1 equivalents). The acetone was stripped and the aqueous residueadjusted to pH 5.8 with dilute HCl. The resulting solution of titleproduct was employed without isolation in the next step.

PREPARATION 7

N-alpha-Methyl-N-alpha-[N-t-butoxycarbonyl)-phenylalanyl-N(imidazole)-(t-butoxycarbonyl)histidine

The entire solution of product from the preceding Preparation wasdiluted to 30 ml. with H₂ O and further diluted with 30 ml. dioxane, andcooled to 0°. The pH was adjusted to 11.0 with dilute NaOH anddi(t-butoxycarbonyl)anhydryde [(t-boc)₂ O; 1.61 ml., 0.007 mol, ca. 1.3equivalents) added, and the temperature maintained at 0° for 1 hourwhile maintaining pH 8.5-11.0 by the addition of dilute NaOH. The bathwas removed, an additional 0.5 ml. of the anhydride was added and the pHquickly stabilized at 10.5. The dioxane was stripped and the aqueousresidue washed 2×30 ml. ethyl acetate. The combined organic layers wereback washed 1×30 ml. H₂ O and the wash combined with the originalaqueous layer, covered with 50 ml. fresh ethyl acetate, and the pHadjusted to 1.4 with dilute HCl. The acid ethyl acetate layer wasseparated combined with a further 30 ml. ethyl acetate wash of theacidic aqueous layers, back washed with 30 ml. fresh H₂ O, dried overNa₂ SO₄ and co-stripped with ether to yield title product, 1.9 g.

PREPARATION 8 N-alpha-(2-Benzyl-3-phenylpropionyl)-histidine MethylEster

Under nitrogen 2-benzyl-3-phenylpropionic acid (dibenzylacetic acid, 5.0g., 0.0208 mol) and histidine methyl ester dihydrochloride (4.72 g.,0.0208 mol) were combined in 100 ml. CH₂ Cl₂ and cooled to 0°.Dicyclohexylcarbodiimide (4.29 g., 0.0208 mol) and thenN-methylmorpholine (4.58 ml., 0.0416 mol) were added and the mixturestirred, allowed to warm to ambient temperature and stirred for 16hours. Insolubles were removed by filtration, filtrate washed 2×100 H₂O, 2×100 ml. saturated NaHCO₃ and 1×100 ml. brine, dried over MgSO₄,stripped to a residue weighing 6.8 g., and the residue chromatographedon 600 g. of silica gel with 99:1 CHCl₃ :CH₃ OH as eluant to producepurified title product, 4.5 g.; ¹ H-nmr (CDCl₃) delta includes delta 3.8(s, 3H, OCH₃).

PREPARATION 9N-alpha-(2-Benzyl-3-phenylpropionyl)-N(imidazole)-(t-butoxycarbonyl)histidine

Title product of the preceding Preparation (4.5 g., 0.0108 mol) and K₂CO₃ (4.66 g., 0.0378 mol) were combined in 25 ml. CH₃ OH and 8 ml. H₂ Oand stirred 16 hours. The methanol was stripped and the aqueous residuediluted with 25 ml. dioxane and 15 ml. of H₂ O. Di-(t-butyl)dicarbonate(2.8 g., 0.013 mol) and the mixture stirred 16 hours. The dioxane wasstripped and the basic aqueous residue extracted with 20 ml. ether,adjusted to pH 2.5 with 5% HCl and extracted 30 ml. of fresh ether. Thelatter organic layer was washed with brine, dried over MgSO₄, stripped,the residue triturated with 15 ml. 1:1 ether:hexane and filtered toyield title product, 1.6 g.; ¹ H-nmr (CDCl₃) includes delta 1.6 (s, 9H,C(CH₃)₃).

We claim:
 1. A compound of the formula ##STR22## wherein R¹⁶ is (C₁-C₆)alkyl, (C₁ -C₆)alkenyl, phenyl, naphthyl, (C₄ -C₇)cycloalkyl, (C₄-C₇)cycloalkenyl, (C₇ -C₉)phenylalkyl, (C₁₁ -C₁₃)naphthylaklyl, (C₅-C₁₀)-(cycloalkyl)alkyl, or one of said groups mono or disubstituted onthe aromatic ring with the same or different groups selected from (C₁-C₃)alkyl, (C₁ -C₃)-alkoxy, fluoro or chloro.
 2. A compound of claim 1wherein R¹⁶ is cyclohexylmethyl.